EA042294B1 - ULTRAPURE AGONISTS OF GUANYLATE CYCLASE C, METHOD FOR THEIR PRODUCTION AND USE - Google Patents

Description

Related Applications

The present application claims the priority and benefits of USSN Provisional Application 61/831402, filed June 5, 2013, the entire contents of which are incorporated herein by reference.

The field of technology to which the invention belongs

The present invention relates to a method for the purification of peptide agonists of guanylate cyclase C, which can be used to obtain compositions for the treatment and prevention of various diseases and disorders.

State of the art prior to the invention

Guanylate cyclase C is a transmembrane form of guanylate cyclase that is expressed on various cells, including epithelial cells of the gastrointestinal tract (reviewed in Vaandrager 2002 Mol. Cell. Biochem. 230:73-83). It was originally discovered as a gut receptor for thermostable toxin (ST) peptides secreted by enterobacteria and causing diarrhea. ST peptides share a primary amino acid structure with two peptides isolated from intestinal and urinary mucosa, guaniline and uroguaniline (Currie, et al., Proc. Nat'l Acad. Sci. USA 89:947-951 (1992); Hamra, et al. al., Proc. Nat'l Acad. Sci. USA 90:10464-10468 (1993); Forte, L., Reg., Pept. 81:25-39 (1999); Schulz, et al., Cell 63: 941-948 (1990); Guba, et al., Gastroenterology 111:1558-1568 (1996); Joo, et al., Am. J. Physiol. 274:G633-G644 (1998)).

In the intestines, guaniline and uroguaniline act as fluid and electrolyte balance regulators. In response to high oral salt intake, these peptides are released into the intestinal lumen, where they bind to guanylate cyclase C located on the luminal membrane of enterocytes (simple columnar epithelial cells of the small intestine and colon). The binding of guaniline peptides to guanylate cyclase C induces the excretion of electrolytes and water into the intestinal lumen through a complex intracellular signaling cascade initiated by an increase in the level of cyclic guanosine monophosphate (cGMP).

The cGMP-mediated signaling initiated by guaniline peptides is critical for normal gut function. Any anomaly in this process can lead to gastrointestinal disorders such as irritable bowel syndrome (IBS) and inflammatory bowel disease. Inflammatory bowel disease is the general name for a group of disorders that cause inflammation of the intestine, characterized by redness and swelling of the tissue. Examples include ulcerative colitis and Crohn's disease. Crohn's disease is a serious inflammatory disease that predominantly affects the ileum and colon, but can also occur in other parts of the gastrointestinal tract. Ulcerative colitis is an inflammatory disease exclusively of the large intestine, large intestine. Unlike Crohn's disease, in which all layers of the intestine are affected, and in which normal, healthy sections of the intestine may be present between the affected areas of the intestine, ulcerative colitis only affects the deepest layers of the lining (mucosa) of the large intestine in a continuous manner. Depending on which part of the gastrointestinal tract is affected, Crohn's disease can be referred to as ileitis, regional enteritis, colitis, etc. Crohn's disease and ulcerative colitis are different from spastic colon or irritable bowel syndrome, which are disorders of the motility of the gastrointestinal tract. Inflammation of the gastrointestinal tract can be a chronic condition. It is estimated that as many as 1,000,000 Americans are affected by inflammatory bowel disease, apparently with an equivalent effect on male and female patients. Most cases are diagnosed before the age of 30, but the disease can occur in the sixth, seventh and later decades of life.

IBS and chronic idiopathic constipation are pathological conditions that can cause severe intestinal discomfort and dysfunction, however, unlike inflammatory bowel disease, IBS does not cause severe inflammation or changes in intestinal tissue and is not thought to increase the risk of colorectal cancer. Previously, inflammatory bowel disease, celiac disease, and IBS were considered completely different disorders. At present, the description of inflammation, albeit minor, in IBS and the overlap of symptoms between IBS and celiac disease call this claim into question. Acute bacterial gastroenteritis is the strongest risk factor identified so far for the subsequent development of post-infectious irritable bowel syndrome. Clinical risk factors include prolonged acute distress and absence of vomiting. Genetically determined sensitivity to inflammatory stimuli may also be a risk factor for irritable bowel syndrome. The underlying pathophysiology includes increased intestinal permeability and non-specific inflammation, as well as altered motility and visceral sensitivity. Serotonin (5-hydroxytryptamine [5-HT]) is a key modulator of gut function and is known to play a key role in the pathophysiology of IBS. 5-HT activity is regulated by cGMP.

While the exact causes of IBS and inflammatory bowel disease (IBD) are not known,

- 1 042294 violation of the process of constant renewal of the mucous membrane of the gastrointestinal tract can contribute to the pathology of the disease in IBD and exacerbate IBS. The process of renewal of the lining of the gastrointestinal tract is an efficient and dynamic process involving the continuous proliferation and accumulation of unwanted damaged cells. Proliferation rates of cells lining the mucosa of the gastrointestinal tract are very high, second only to the hematopoietic system. Homeostasis of the gastrointestinal tract depends on both proliferation and programmed cell death (apoptosis) of epithelial cells lining the mucosa of the gastrointestinal tract. Cells are continuously shed from the villi into the intestinal lumen and are replenished at a substantially equal rate through cell proliferation in the crypts, followed by their upward movement to the villi. Cell proliferation and apoptosis rates in the intestinal epithelium can increase or decrease under a variety of conditions, for example, in response to physiological stimuli such as aging, inflammatory signals, hormones, peptides, growth factors, chemicals, and dietary habits. In addition, an increased rate of proliferation is often associated with a decrease in renewal time and an expansion of the zone of proliferation. The proliferation index is much higher in pathological conditions such as ulcerative colitis and other disorders of the gastrointestinal tract. Intestinal hyperplasia is the main stimulant of inflammation of the gastrointestinal tract. Apoptosis and cell proliferation together regulate the number of cells and determine the proliferation index. Decreased rates of apoptosis are often associated with abnormal growth, inflammation, and neoplastic transformation. Thus, both increased proliferation and/or decreased cell death can increase the intestinal tissue proliferation index, which in turn can lead to inflammatory diseases of the gastrointestinal tract.

In addition to the role of uroguaniline and guaniline as modulators of intestinal fluid and ion secretion, these peptides may also be involved in the continuous renewal of the gastrointestinal mucosa by maintaining a balance between proliferation and apoptosis. For example, the peptides uroguaniline and guaniline appear to stimulate apoptosis by controlling cellular ion flux. Given the prevalence of inflammatory conditions in Western populations, there is a need to improve treatment options for inflammatory conditions, particularly those of the gastrointestinal tract.

Peptide guanylate cyclase C agonists (GCC agonists) are described in US Pat. 7,041,786, 7,799,897 and US Patent Application Publication Nos. US2009/0048175, US2010/0069306, US2010/0120694, US2010/0093635 and US2010/0221329, and WO2012/118972. However, prior synthesis of peptides for pharmaceutical use presents a number of specific problems such as low overall yield (e.g., less than 10%) and/or high levels of impurities (e.g., contaminants derived from organic solvents used during synthesis or purification, and degradation products or topoisomers formed, for example, during purification).

The essence of the invention

In one aspect, the present invention stems from an attempt to overcome various unexpected problems encountered during the purification process of GCC peptide agonists for pharmaceutical use, such as the lyophilization process and the precipitation process described in WO2012/118972. The methods described herein provide a solution to these problems.

In one aspect, the present invention provides a purified peptide comprising a GCC agonist sequence selected from the group consisting of SEQ ID NOs: 1, 9, and 104, wherein the purified peptide has the following characteristics:

a) has a bulk density of not more than 0.1 g/ml;

b) contains less than 50 ppm acetamide;

c) less than 0.3% alpha-Asp-9-plenacanatide.

The purified peptide may have one or more of the following features.

For example, the peptide is stable at 25°C for at least three months.

For example, the peptide has a particle size distribution having a D10 value of about 2 to 15 microns; a D50 value of approximately 15-50 µm; and a D90 value of approximately 40-80 µm as measured by light scattering with a liquid dispersant.

For example, the purified peptide contains no more than 35 ppm of acetamide (eg, <18 ppm).

For example, a purified peptide contains less than 0.15% alpha-Asp-9-plenacanatide (having a relative retention time (RRT) of ˜1.33 as determined by ultra high performance liquid chromatography (HPLC) analysis described herein).

For example, the purified peptide has a bulk density of no more than 0.09 g/ml, no more than 0.08 g/ml, no more than 0.07 g/ml, no more than 0.06 g/ml, no more than 0.05 g/ml ml, not more than 0.04 g/ml or not more than 0.03 g/ml.

For example, the purified peptide is substantially free of water (for example, the water content does not exceed 10, 9, 8, 7, 6, 5, 4, 3.5, 3, 2.5, 2, 1.5, 1 , 0.5, 0.25 or 0.1%, based on the total weight of the peptide).

For example, the purified peptide has a chromatographic purity of at least 95%, at least 96%, or at least 97%.

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For example, the total content of impurities in the purified peptide is less than 3% (eg, <2 or <1%).

For example, the purified peptide is furthermore substantially free of one or more impurities selected from acetonitrile, alcohols, ammonium, acetates, and TFA.

For example, a purified peptide contains less than 300 ppm acetonitrile (eg, <250 ppm).

For example, the purified peptide contains less than 0.2% TFA (eg, <0.15%, <0.1%, <400 ppm, <300 ppm, <200 ppm, <100 ppm, or <50 ppm).

For example, a purified peptide contains less than 0.2% isopropanol, i. e. IPA (e.g. <0.15%, <0.1%, <1000ppm, <900ppm <800ppm, <700ppm, <600ppm, <500ppm, <400ppm, <300ppm, <200ppm , <100 ppm, <50 ppm or <20 ppm).

For example, a purified peptide contains less than 0.25% acetate (eg, <0.2% or <0.1%).

For example, the purified peptide is substantially free of topoisomers (eg, <0.4%, <0.3%, <0.2%, or <0.1%).

For example, the purified peptide is substantially free of iso-Asp2-plecanatide (RRT 0.96-0.97) (e.g., <0.4, <0.3, <0.2, or <0.1%) .

In another aspect, the invention also relates to a method for purifying/isolated a peptide containing a GCC agonist sequence selected from the group consisting of SEQ ID NOs: 1-251. The method includes: obtaining a first peptide solution containing a peptide containing a GCC agonist sequence selected from the group consisting of SEQ ID NOs: 1-251, water and acetonitrile; loading the C18 or polymer adsorbent column with the first peptide solution to adsorb the peptide on the polymer adsorbent column, eluting the peptide from the C18 or polymer adsorbent column with an aqueous alcohol solution to obtain a second peptide solution, reducing the amount of alcohol in the second peptide solution, and lyophilizing the second peptide solution so that a dry peptide is obtained.

The method may include one or more of the following features.

For example, an aqueous alcohol solution contains isopropanol (eg, with an isopropanol content in an aqueous alcohol solution of approximately 40%).

In another embodiment, the aqueous alcohol solution contains propanol, t-butanol, 2-butanol, or ethanol.

For example, the first peptide solution further contains acetamide.

For example, the first peptide solution further contains acetic acid (eg 0.2%) or triethylamine phosphate (eg 1%).

For example, the amount of alcohol (eg isopropanol) in the second peptide solution is reduced, eg by rotary evaporation, to less than 5%.

For example, the method further includes dissolving the dry peptide in water to obtain a third peptide solution after lyophilization. For example, the third peptide solution further contains ammonium acetate or ammonium hydroxide (eg, such that the third solution has a pH of about 5).

For example, the method further includes lyophilizing the third peptide solution such that a purified peptide is obtained.

For example, the peptide in the first peptide solution is prepared by the fragment condensation method (ie, solution-solid phase hybrid method) as described in WO2012/118972. In one embodiment, the first peptide solution is obtained in a salt exchange step in which the peptide is washed with an aqueous solution of acetonitrile containing triethylamine phosphate or acetic acid.

For example, a polymeric adsorbent column is a C18 column for preparative RP-HPLC. In one embodiment, the polymeric adsorbent column contains a polystyrene resin. In particular, the resin is selected such that the eluted or desorbed purified peptide is at least 80% of the amount of peptide adsorbed on the resin, such as at least 85%, at least 90%, or at least 95%. In one embodiment, the resin is formed from cross-linked polystyrene with an average pore diameter greater than 5 nm, such as approximately 6-8 nm, 10-15 nm, 15-20 nm, or 25-30 nm.

In another aspect, the invention also relates to a purified peptide obtained by the purification process of the invention. Purified peptides may have one or more of the following features.

For example, the purified peptide contains a GCC agonist sequence selected from the group consisting of SEQ ID NOS: 1, 9, and 104.

For example, the purified peptide has a chromatographic purity of at least 96%, at least 97%, or at least 98%. For example, the GCC agonist peptide has a chromatographic impurity content of no more than 4%, no more than 3.5%, no more than 3%, no more than 2.5%, no more than 2%, no more than 1.5%, or no more than 1%. The chromatographic content of impurities is determined as the total area percentage for impurities by HPLC. The chromatographic content of impurities includes the content of topoisomers. Impurities do not include any pharmaceutically acceptable excipient used to formulate the drug.

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For example, the purified peptide is substantially free of contaminants resulting from the peptide preparation process, such as organic solvents used in the process, e.g., ammonium, acetonitrile, acetamide, alcohol (e.g., methanol, ethanol, or isopropanol), TFA, ether or other contaminants. In this context, substantially free of contaminants means that the peptide contaminant content at the end of the purification process is preferably less than 0.5%, less than 0.3%, less than 0.25%, less than 0.1%, less than 0.05%. , less than 0.04%, less than 0.03%, less than 0.02%, less than 0.01%, less than 0.005%, less than 0.003% or less than 0.001% of the total mass of the peptide. For example, the purified peptide contains <50 ppm acetamide (e.g., <35 or <18 ppm), <300 ppm acetonitrile (e.g., <250 ppm), <1000 ppm TFA (e.g., <400, <300, <200, <100 or <50 ppm), <2000 ppm isopropanol (e.g., <1500, <1000, <500, <400, <300, <200, <100, <50, or <20 ppm), and/or <0.25% acetate (eg, <0.2 or <0.1%). The content of contaminants can be determined by conventional methods such as gas chromatography. Preferably, the amounts of residual solvents in the purified peptide of the invention are less than the limits set forth in the ICH guidelines, for example, IMPURITIES: GUIDELINE FOR RESIDUAL SOLVENTS Q3C(R5) (available at http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/ Guidelines/Quality/Q3C/Step4/Q3C_R5_Step4.pdf).

For example, the purified peptide contains less than 0.3% (eg, <0.15%) alpha-Asp-9plenacanatide (RRT 1.33).

For example, the purified peptide has a bulk density of no more than 0.09 g/ml, no more than 0.08 g/ml, no more than 0.07 g/ml, no more than 0.06 g/ml, no more than 0.05 g/ml ml, not more than 0.04 g/ml or not more than 0.03 g/ml.

For example, the purified peptide is essentially free of iso-Asp2-plecanatide (RRT~0.96-0.97). In this context, substantially free of iso-Asp2-plecanatide means that the content of iso-Asp2-plecanatide in the peptide at the end of the purification process is preferably less than 2%, less than 1.5%, less than 1.25%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2% or less than 0, 1%, based on the total weight of the peptide.

For example, the purified peptide is substantially free of topoisomers. In this context, substantially free of topoisomers means that the topoisomer content of the peptide at the end of the purification process is preferably less than 2%, less than 1.5%, less than 1.25%, less than 1%, less than 0.9%, less than 0. 8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1%, of the total peptide weight .

For example, the purified peptide is substantially free of water. In this context, essentially free from water means that the water content of the peptide at the end of the purification process is preferably less than 10%, 9%, 8%, 7%, less than 6%, less than 5%, less than 4.5%, less than 4 .25%, less than 4%, less than 3.5%, less than 3%, less than 2.5%, less than 2%, less than 1.5%, less than 1%, less than 0.5%, less than 0.25% or less than 0.1%, based on the total weight of the peptide.

For example, the peptide has a particle size distribution having a D10 value of about 2 to 15 microns; a D50 value of approximately 15-50 µm; and a D90 value of approximately 40-80 µm as measured by light scattering with a liquid dispersant.

For example, the purified peptide has a particle size distribution characterized by a D50 value of approximately 600 µm as measured by light scattering with an air dispersant. In comparison, peptides purified by the lyophilization and precipitation methods described in WO2012/118972 have D50 values of approximately 180-250 μm and approximately 300 μm, respectively.

For example, a purified peptide produced by the methods of the invention has an acceptable size distribution for a pharmaceutical formulation. In one embodiment, the peptide (e.g., SP-304) has a size distribution (e.g., average size 80-120 µm) comparable to the size distribution of a pharmaceutical excipient (e.g., microcrystalline cellulose) used in a formulation, e.g., in a unit dosage form. form of 3 mg/day. The size distribution of the purified peptide may vary based on a single dose. For example, when a single dose is less than 3 mg/day, the purified peptide in the pharmaceutical formulation has a smaller average size than at a dose of 3 mg/day. For example, a purified peptide produced by the methods of the invention is milled to achieve an acceptable size distribution.

The size distribution of the peptide of the invention can be determined by conventional methods such as sieve analysis, shading or dynamic light scattering assays.

The invention also relates to a formulation (e.g., oral formulation) containing peptides prepared and/or purified by the methods described herein, and in particular to a low dose formulation containing 0.05-10 mg (e.g., 0.1 mg, 0.3 mg or 0.5 mg) of purified peptides. The low dose formulation may further have one or more additional features as described in WO2012/037380 and US2012-0237593 and may be prepared by the methods described herein such as dry blending.

Other features and advantages of the invention will be apparent from the following detailed description and claims, within the scope of which they are included.

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Brief description of the drawings

Fig. 1 is a graph showing the particle size distribution by sieve analysis for lyophilized plecanatide and precipitated plecanatide.

Fig. 2 is an optical microscopy image of lyophilized plecanatide.

Fig. 3 is an optical microscopy image of precipitated plecanatide.

Fig. 4 is an HPLC chromatogram of plecanatide isolated according to one embodiment of the purification method of the invention.

Detailed description

In one aspect, the present invention relates to solving various unexpected problems encountered during the purification processes of GCC peptide agonists for pharmaceutical use, such as the lyophilization process and the precipitation process described in WO2012/118972.

In particular, it was found that the lyophilization method described in WO2012/118972, involving the use of an acetonitrile/water solvent, unexpectedly resulted in an enrichment of residual acetamide (acetonitrile trace impurities) in the acetamide-rich lyophilized plecanatide product (i.e. ranging from 88 to 453 ppm among batches tested, or approximately 300 ppm on average), which prevented the commercialization of plecanatide at doses greater than 3 mg/day. In addition, the lyophilized plecanatide product also had high levels of residual salts such as TFA, acetate, and ammonium salts.

On the other hand, while the plecanatide product purified by the precipitation method described in WO2012/118972 had a low residual acetamide content (<50 ppm) and a higher bulk or bulk DENSITY (i.e. about ten times higher), than the lyophilized product, the precipitated plecanatide product contained high levels of residual solvents (eg, IPA as high as 90,000 ppm) due to the difficulty in removing the solvents used to precipitate the peptide. In addition, while heating to low temperature (45°C) during vacuum drying helped to reduce the amount of residual isopropanol to below the ICH limit of 5000 ppm (for example, 1700 ppm), the thermal degradation product, alpha-Asp-9-plecanatide , having a relative retention time (RRT) of ~1.33 in an ultra performance liquid chromatography (HPLC) assay, was then formed in the final peptide product at a concentration as high as 0.9%. The RRT 1.33 impurity content also increases in plecanatide and its tablets during storage at room temperature. Thus, the initial high level of this RRT impurity of 1.33 in plecanatide produced by precipitation results in a smaller acceptance window and thus shortens the usable shelf life of plecanatide drug products.

The purification methods of the invention address the problem of residual solvent and degradation, for example, by replacing the acetonitrile/water solvent with the IPA/water solvent, followed by rotary evaporation to reduce the IPA level to a critically low level (e.g., <5%), followed by first partial lyophilization resulting in low but unacceptable levels of residual solvents, followed by dilution in water and final lyophilization resulting in low and acceptable levels of residual solvents for pharmaceutical use. In addition, the purification methods of the invention are suitable for scale-up production/purification of peptides.

In addition, it has surprisingly been found that the addition of a small amount of ammonium acetate buffer (eg 0.5% of dry peptide) during dilution with water (pH 5) prior to final lyophilization improves the solubility of the final product. In addition, ammonium acetate buffer (pH 5) reduces the rate of topo-isomerization of plecanatide in solution during lyophilization. The ammonium acetate buffer (pH 5) also controls the levels of residual salts in the final product with lower diversity compared to the lyophilization product described in WO2012/118972.

The peptide at the end of the purification or isolation processes of the invention has a low level of residual acetamide (<50 ppm) and a bulk density of not more than 0.1 g/ml, as well as a lower total level of residual solvents (for example, <500 ppm IPA, <300 ppm ACN), low levels of impurities due to degradation (eg, <0.1% degradation product of alpha-Asp-9-plecanatide (RRT 1.33)) and topoisomers (eg, 0.4% or less), and low levels residual salts (eg <0.1% TFA, 0.08-0.23% acetate and 0.11-0.17% ammonium). A comparison between plenacanatide products isolated by various methods is presented in Table 1. XX below.

The invention relates to methods for purifying or isolating peptides, for example GCC peptide agonists, in particular peptides obtained by a solution-solid phase hybrid process, for example as described in WO2012/118972, the contents of which are hereby incorporated by reference. The hybrid method includes obtaining two or more fragments of the peptide of interest through solid phase and/or solution phase synthesis and combining them through solution phase synthesis to obtain the target peptide. The method may further include, if necessary, oxidative cyclization of the cysteine amino acid residues of the linear peptide obtained by connecting the fragments to obtain a cyclized peptide.

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The fragments described above can be prepared by conventional liquid phase peptide synthesis or solid phase peptide synthesis methods in which the peptide bond is formed by direct condensation of the amino group (i.e. NH2) of the first amino acid with the carboxy group (i.e. COOH) of the second amino acid with the exception of water molecules. In one embodiment, at least one of the fragments is obtained by solid phase peptide synthesis.

Synthesis of a peptide bond by direct condensation, as formulated above, requires suppression of the reactive nature of the amino group of the first and carboxyl group of the second amino acid. Masking substituents (ie, protecting groups) should allow easy removal without inducing degradation of the labile peptide molecule.

The term protected peptide or protected peptide fragment refers to a peptide or peptide fragment in which all reactive groups on their constituent amino acids are masked by protective groups, unless otherwise indicated. The term deprotected peptide or deprotected fragment of a peptide refers to a peptide or fragment of a peptide in which all reactive groups on their constituent amino acids are unprotected from masking, unless otherwise indicated. The term reactive groups refers to groups that form a peptide bond and to groups that interfere with the formation of a peptide bond, such as amino, carboxyl, hydroxyl, and thiol groups (as in cysteine). Examples of amino protecting groups include, and are not limited to, 9-fluorenylmethyloxycarbonyl (Fmoc), t-butoxycarbonyl (Boc), benzoyl (Bz), acetyl (Ac) and benzyl (Bn). Examples of carboxyl protecting groups include trityl (triphenylmethyl, Trt) and O-tert-butyl (OtBu). Examples of thiol protecting groups include acetamidomethyl (Acm), tert-butyl (tBu), 3-nitro-2-pyridinesulphenyl (NPYS), 2-pyridinesulphenyl (Pyr), and trityl (Trt). Additional examples of protecting groups are described in Greene, TW, Wuts, PGM, Protective Groups in Organic Synthesis, ^3rd edition, John Wiley & Sons: New York, 1999, the contents of which are incorporated herein by reference.

For example, hybrid synthesis methods have been used to prepare SP-304 (plecanatide). Specifically, three peptide fragments, A, B and C, are obtained, and then a linear peptide sequence is assembled by condensing fragments A, B and C as follows: preparation of fragment A, Boc-Asn(Trt)-Asp(OtBu)-Glu(OtBu )-Cys(Trt)-Glu(OtBu)-Leu-OH, via solid phase synthesis on 2chlorotrityl chloride resin; obtaining fragment B, Fmoc-Cys(Acm)-Val-Asn-Val-Ala-Cys(Trt)Thr(tBu)-Gly-OH, by solid phase synthesis on 2-chlorotrityl chloride resin; obtaining fragment C, Cys(Acm)-Leu-OtBu, by liquid phase synthesis, combining fragments B and C in liquid phase to obtain a fragment B-C, and combining fragments A and B-C to obtain a linear peptide A-B- WITH.

Side chain protected fragments A (BocAA1-6OH) and B (FmocAA7-14OH) can be generated by Fmoc SPPS using ultra-acid-sensitive 2-chlorotrityl chloride (2ClTrt) resin and Fmoc-protected amino acid derivatives as shown in Scheme 1 below.

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Scheme 1.

2-;

2-;

Fmoc-Leu-OH

DIEAb DMF/DCM

DBU, HOBt NHFmoc piperidine

i) Peptide chain assembly using Fmoc chemistry ii) 1%TFA/DCM

Fmoc-Gly-OH

DIEA in DMF/DCM

DBU, HOBt piperidine

Fmoc-Cys(Acm)-Val-Asn(Trt)-Val-Ala-Cys(Trt)-ThrttBu)-Gly-OH, (Fragment B or FmocAA7-14OH)

Fragment C (HAA15-16OtBu) can be obtained via liquid phase synthesis and then combined with fragment B (FmocAA7-14OH) in liquid phase to obtain fragment B-C (FmocAA7-16OtBu). The Fmoc protecting group can then be removed from fragment B-C (FmocAA7-16OtBu) to give HAA7-16OtBu, which is then coupled to fragment A (BocAA1-6OH) to give linear SP304 with side chain protection (BocAA1-16OtBu) as shown in Figure diagram 2 below.

Scheme 2.

H-Leu-OtBu.HCI

DIEA, HOBt, HBTU + Fmoc-Cys(Acm)-OH—-----*

Fmoc-Cys(Acm)-Leu-OtBu FmocAA15-160tBu

Piperidine/DCM

H-Cys(Acm)-Leu-OtBu HAA15-16OtBu

DIEA, CI-HOBt, HBTU

Piperidine/DCM

FmocAA7-14OH, DMF

FmocAA7-160tBu

DIEA, CI-HOBt, HBTU

HAA7-16OtBu --------------*

BgcAA1-6OH,

DMF

BocAA1-16OtBu

TIS/EDT/TFA

HAA1-16OH

H-Asn-Asp-Glu-Cys-Glu-Leu-Cys(Acm)-Val-Asn-Val-Ala-Cys-Thr-Gly-Cys(Acm)-Leu-OH

Linear side chain protected SP-304 (BocAA1-16OtBu) can be treated with trifluoroacetic acid/triisopropylsilane/ethanedithiol (TFA/TIS/EDT) to form partially protected SP-304 (HAA1-16OH) which has 2 S-Acm groups (as shown in Scheme 2 above) are intact. Partially protected linear SP-304 (HAA1-16OH) can be oxidized with H ₂ O ₂ , followed by simultaneous removal of the S-Acm groups and disulfide formation with iodine to give crude dicyclic SP-304 as shown in Schemes 3 and 4 below.

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Scheme 3.

H-Asn-Asp-Glu-Cys-Glu-Leu-Cys(Acm)-Val-Asn-Val-Ala-Cys-Thr-Gly-Cys(Acm)-Leu-OH

NAA1-16OH I n ₂ O ₂

H-Asn-Asp-Glu-Cys-Glu-Leu-Cys(Acm)-Val-Asn-Val-Ala-Cys-Thr-Gly-Cys(Acm)-Leu-QH monocyclized HAA1-16OH

II ₂ /AcOH

H-Asn-Asp-Glu-(j:ys-Glu-Leu-(i)ys-Val-Asn-Val-Ala-Cys-Thr-Gly-(J;ys-Leu-OH dicyclized HAA1-16OH | Application on a column with resin from a polystyrene adsorbent | Elution with ethanol

G I

H-Asn-Asp-Glu-(j)ys-Glu-Leu-Cys-Val-Asn-Val-Ala-Cys-Thr-Gly-Cys-Leu-OH dicyclized HAA1-16OH

Scheme 4.

H-Asn-Asp-Glu-Cys-Glu-Leu-Cys(Acm)-Val-Asn-Val-Ala-Cys-Thr-Gly-Cys(Acm)-Leu-OH

NAA1-16ON | _H2 O ₂

H-Asn-Asp-Glu-Cys-Glu-Leu-Cys(Acm)-Val-Asn-Val-Ala-Cys-Thr-Gly-Cys(Acm)-Leu-OH monocyclized HAA1-16OH j I ₂ /AcOH

H-Asn- Asp-Glu-Cys-Glu- Leu-iys-Val-Asn-Val-Ala-Cys-Thr-Gly-(!ys- Leu-0 Η dicyclized HAA1-16OH j Deposition on a RP-HPLC column j Elution with ACN/H2O j Pooling of fractions of suitable quality j Salt exchange and lyophilization

H- As η -A sp -GI u-(^ys-Glu-Leu-^ys-Val-Asn-Val-Ala-Cys-Thr-Gly-C y s-Le u-0 H

Medicinal substance SP 304

The crude dicyclic SP-304 solution can then be purified and concentrated as shown in Scheme 3 above by applying the solution to a polystyrene adsorbent resin column (for example, D101 (Anhui Sanxing (China); cross-linked polystyrene; surface area 500-550 m ² /g; average pore diameter: 9-10 nm; pore volume: 1.18-1.24 ml/g; bulk density: 0.65-0.70 g/ml; specific gravity: 1.03-1. 07g/ml Moisture: 67-75% Particle Size: 0.315~1.25mm >95% Effective Diameter: 0.4~0.7mm Uniformity Ratio: <1.6%), DA201C, DA201H , ADS-8, and ADS-5), eluting the dicyclic SP-304 from the column with an eluent (e.g., 90% aqueous ethanol), concentrating the collected SP-304 solution under reduced pressure, and precipitating the SP-304 with methyl tert- butyl ether (MTBE). The precipitate can then be collected by filtration or centrifugation, dried under high vacuum to give SP-304 in solid form.

As illustrated in Scheme 4 above, a solution of crude dicyclic SP-304 can also be purified directly on a C18 preparative HPLC column with acetonitrile (ACN), methanol and/or water in various buffer systems. Crude dicyclic SP-304 can also be purified by other methods known to those skilled in the art.

Those skilled in the art will recognize that in solid phase synthesis, deprotection and reactions

- 8 042294 binding must go to completion, and blocking groups for the side chains must be stable throughout the synthesis.

N-terminal acetylation can be carried out by reacting the final peptide with acetic anhydride prior to cleavage from the resin. C-amidation is carried out using a suitable resin such as methylbenzhydrylamine resin using Boc technology.

When synthesized in the liquid phase, a wide variety of coupling methods and protecting groups can be used (see, Gross and Meienhofer, eds., The Peptides: Analysis, Synthesis, Biology, Vol. 1-4 (Academic Press, 1979); Bodansky and Bodansky , The Practice of Peptide Synthesis, 2d ed. (Springer Verlag, 1994)). In addition, intermediate cleaning and linear scaling are possible. Those skilled in the art will recognize that solution synthesis requires consideration of the protecting groups for the backbone and side chains, and the mode of activation. In addition, careful selection of fragments is required to minimize racemization during fragment condensation. For example, racemization is minimized when the fragments contain a C-terminal Gly or Pro. Solubility is also considered as a factor. Solid phase peptide synthesis uses an insoluble polymer to support organic synthesis. The polymer-supported peptide chain allows the use of simple washing and filtration steps instead of time-consuming purification in intermediate steps. Solid phase peptide synthesis can generally be carried out according to the method of Merrifield et al., J. Am. Chem. Soc, 1963, 85:2149, which involves the assembly of a linear peptide chain on a resin support using protected amino acids. Solid phase peptide synthesis typically uses either Boc or Fmoc methods, both of which are well known in the art.

The general hybrid synthesis of SP-353 involves solid phase and liquid phase synthesis to obtain suitable peptide fragments (see Schemes 5 and 6 below), subsequent condensation of the fragments to obtain a linear crude peptide (see Scheme 7 below), and natural oxidative folding to obtain a cyclized end product (see scheme 7 below). The same method can also be used to produce other ST peptide analogs (such as SP-354, linaclotide, etc.) with similar amino acid sequences shown in Table 1. II.

Scheme 5.

- 9 042294

Scheme 6.

Solid-phase synthesis of a C-terminal dipeptide with side chain protection

About OtBu

H-Tyr(tBu)-OtBu.HCI Fmoc-Cys(Trt)-OH _{15 1R}

H-Cys ¹⁵ (Trt)-Tyr ¹⁶ (tBu)OtBu

i) HOBt, HBTU, DIPEA, DMF;

ii) 5% piperidine in DCM ^H ' ^15-16OtBu

Scheme 7.

Fragment condensation, disulfide bridge folding and purification

H-Cys ¹⁵ (Trt)-Tyr ¹⁶ (tBu)-aBu + Fmoc-Ala ¹¹ -Cys ¹² (Trt)-Thr ¹³ (tBu)-Gly ¹⁴ -OH

H-AA15-160tBu Fmoc-AA11-14OH

IHBTU, CI-HOBt, DIPEA DMF

Fmoc-Ala ¹¹ -Cys ¹² (Trt) -Thr ¹³ (tBu) -Gly ¹⁴ -Cys ¹⁵ (Trt) -Tyr ¹⁶ (tBu) -OtBu

Fmoc-AA11-16 OtBu

J 5% piperidine in DMF

H-Ala ¹¹ -Cys ¹² (Trt) -Thr ¹³ (tBu) -Gly ¹⁴ -Cys ¹⁵ (Trt) -Tyr ¹⁶ (tBu) -OtBu

H-AA11-16OtBu

HBTU, CI-HOBt, DI PE A (Boc-AA1-10OH,DMF

Boc-A3n ¹ (Trt)-Phe ² -Cys ² (Trt)-Cys ⁴ (Trt)-Glu ^E (OtBu)-Ser(tBu)-Cys ⁷ (Trt)-Cys ⁸ (Trt)-Asn ^& iTrt) -Pro ¹⁰ -Aa ¹¹ -Cys ¹² (Trt)

T hr ¹³ (tB u)-GI y%C ys ¹⁵ (Trt)- Tyr ¹⁶ (t Bu)- Ot Bu | TFA:EDT:TIS:H ₂ O (85:5:5:5,v/v/v/v), 2h

H-Asn ¹ -Phe ² -Cys ³ -Cys ⁴ -Glu ⁵ -Ser ⁶ -Cys ⁷ -Cys ⁸ -Asn ⁹ -Pro ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Tyr ¹⁶ - Oh

Oxidative folding I 0.1 M NaHCO ₃ , 1 M GuHCI, 3 mM cysteine and 0.3 M cystine, pH 8.1, * in 15% aqueous ACN

H-Asn ¹ -Phe ² -Cys ³ -Cys ⁴ -Glu ⁵ -Ser ⁶ -Cys ⁷ -Cys ⁸ -Asn ⁹ -Pro ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cy^

Preparative RP-HPLC, Counterion exchange, ,, Lyophilization

Medicinal substance SP-353

The general method for hybrid synthesis of SP-333 includes solid phase and liquid phase synthesis to obtain suitable peptide fragments (see Schemes 8 and 9 below), subsequent condensation of the fragments to obtain a linear crude peptide (see Scheme 9 below), oxidative folding to obtain a cyclized end product, purification and lyophilization (see scheme 10 below).

- 10 042294

Scheme 8.

Scheme 9.

DIEA, HOBt, HBTU

HD-Leu-OtBu.HCI + Fmoc-Cys(Acm)-OH———-----► Fmoc-Cys(Acm)-D-Leu-OtBu ^DMF FmocAA15-16OtBu

Piperidine/PCM* H-Cys(Acm)-D-Leu-OtBu ^DIEA ' Ο-HOBt, HBTU HAA15-160tBu DMF ^CAA7 ' ^14OH ' FmocAA7-16OtBu

Piperidine/DCM DIEA, CI-HOBt, HBTU TIS/EDT/TFA

HAA7-160tBu --------------* BocAAl-160tBu -----► HAA1-16OH

BocAA1-6OH,

DMF

H-D-Asn-Asp-Glu-Cys-Glu-Leu-Cys(Acm)-Val-Asn-Val-Ala-Cys-Thr-Gly-Cys(Acm)-D-Leu-0 H

- 11 042294

Scheme 10.

H-D-Asn-Asp-Glu-Cys-Glu-Leu-Cys(Acm)-Val-Asn-Val-Ala-Cys-Thr-Gly-Cys(Acm)-D-Leu-OH

NAA1-16ON _n η

H-D-Asn-Asp-Glu-Cys-G lu-Leu-Cys(Acm)-Val-Asn-Val-Ala-Cys-Thr-Gly-Cys(Acm)-D-Leu-OH monocyclized HAA1-16OH

II ₂ /AcOH

H-D-Asn-Asp-Glu-Cys-Glu-Leu-Cys-Val-Asn-Val-Ala-Cys-Thr-Gly-Cys-D-Leu-OH I________________________________________________________________________________________________________________________________________________________________________________________________________________________________ I dicyclized HAA1-16OH | Loading onto a RP-HPLC column j Elution with ACN/HgO

Pooling of fractions of suitable quality

I Salt exchange and lyophilization

H-D-Asn-Asp-Glu-Cys-Glu-Leu-Cys-Val-Asn-Val-Ala-Cys-Thr-Gly-Cys-D-Leu-OH

Medicinal substance SP 333

A general method for purifying peptides, including peptides synthesized by the hybrid methods described herein, includes, for example, the steps illustrated in Scheme 11 below. It should be understood that specific steps in Scheme 11 may be repeated (eg, washing the column with deionized water) or omitted (eg, salt exchange or removal of alcohol after dehydration) to optimize the purification method.

Scheme 11.

Peptide, such as dicyclized SP-304 or SP-333 | Application to a RP-HPLC column

Elution with ACN/HgO j Pooling of fractions of suitable quality, eg >95% HPLC purity j Salt exchange | Pooling of fractions of suitable quality, e.g. >95% HPLC purity

I Application of the main pool on a column packed with a polymeric adsorbent * for desalting y Washing the column with deionized water

I Elution of the peptide with an aqueous solution of alcohol (e.g. isopropanol/water) and Collection of the eluate

Removal of water, e.g. by azeotropic distillation

Removal of alcohol, e.g. under reduced pressure

I Addition of an ether to an anhydrous peptide, e.g. cold diethyl ether

Drying the peptide in vacuum

Purified peptide for further drug manufacturing process

The precipitation method as illustrated in Scheme 11 produces a purified peptide, eg GCC peptide agonists. Preferably, the purified peptide is SP-304 (SEQ ID NO: 1) or SP-333 (SEQ ID NO: 9). In one embodiment, purified SP-304 or SP

- 12 042294

333 has a bulk density of at least 0.05 g/ml, at least 0.1 g/ml, at least 0.2 g/ml, at least 0.3 g/ml, at least 0.4 g/ml, or not less than 0.5 g/ml. In a preferred embodiment, purified SP-304 or SP-333 has a bulk density of about 0.05-2 g/ml, about 0.2-0.7 g/ml, about 0.3-0.6 g/ml, or approximately 0.4-0.5 g/ml. In one embodiment, purified SP-304 or SP-333 has a bulk density of at least 0.08 g/ml, at least 0.1 g/ml, at least 0.15 g/ml, at least 0.2 g /ml, not less than 0.3 g/ml, not less than 0.4 g/ml, not less than 0.5 g/ml or not less than 0.6 g/ml.

For example, purified SP-304 or SP-333 has a bulk density of 0.08-2 g/ml, about 0.4-0.9 g/ml, about 0.5-0.8 g/ml, or about 0.6 -0.7 g/ml. In one embodiment, the purified SP-304 or SP-333 peptide contains <0.01% acetamide (e.g., <28 ppm), <0.3% ammonium ion (e.g., <0.25%), <0. 01% acetonitrile (eg <20 ppm), and/or <0.1% TFA (eg <0.09%). In one embodiment, the purified SP-304 or SP-333 peptide has a bulk density of 0.4-0.5 g/mL, has a bulk density of 0.6-0.7 g/mL, and contains <0.01% acetamide (e.g., <28 ppm), <0.3% ammonium ion (e.g., <0.25%), <0.01% acetonitrile (e.g., <20 ppm), and/or <0.1% TFA (e.g., <0.09%).

Some embodiments provide a purified peptide having a GCC agonist sequence selected from the group consisting of SEQ ID NOs: 1, 9, and 104, wherein the purified peptide has the following characteristics:

a) has a bulk density of not more than 0.1 g/ml;

b) contains less than 50 ppm acetamide;

c) less than 0.3% alpha-Asp-9-plenacanatide.

The purified peptide may have one or more of the following features.

In one embodiment, the peptide is stable at 25°C for at least three months.

In one embodiment, the peptide has a particle size distribution having a D10 value of about 2 to 15 microns; a D50 value of approximately 15-50 µm; and a D90 value of approximately 40-80 µm as measured by light scattering with a liquid dispersant.

In one embodiment, the purified peptide contains no more than 35 ppm acetamide (eg, <18 ppm).

In one embodiment, the purified peptide contains less than 0.15% alpha-Asp-9 plenacanatide (having a relative retention time (RRT) of ∼1.33 by ultra high performance liquid chromatography (HPLC) analysis described herein).

In some embodiments, the purified peptide has a bulk density of at most 0.09 g/ml, at most 0.08 g/ml, at most 0.07 g/ml, at most 0.06 g/ml, at most 0, 05 g/ml, not more than 0.04 g/ml, or not more than 0.03 g/ml.

In some embodiments, the purified peptide is substantially free of water (e.g., the water content does not exceed 10, 9, 8, 7, 6, 5, 4, 3.5, 3, 2.5, 2, 1, 5, 1, 0.5, 0.25 or 0.1%, based on the total weight of the peptide).

In some embodiments, the purified peptide has a chromatographic purity of at least 95%, at least 96%, or at least 97%.

In some embodiments, the implementation, the total content of impurities in the purified peptide is less than 3% (for example, <2 or <1%).

In some embodiments, the purified peptide is furthermore substantially free of one or more impurities selected from acetonitrile, alcohols, ammonium, acetates, and TFA.

For example, a purified peptide contains less than 300 ppm acetonitrile (eg, <250 ppm).

In some embodiments, the purified peptide contains less than 0.2% TFA (eg, <0.15%, <0.1%, <400 ppm, <300 ppm, <200 ppm, <100 ppm, or <50 ppm).

In some embodiments, the purified peptide contains less than 0.2% isopropanol, i. IPA (e.g. <0.15%, <0.1%, <1000ppm, <900ppm, <800ppm, <700ppm, <600ppm, <500ppm, <400ppm, <300ppm, <200 ppm, <100 ppm, <50 ppm, or <20 ppm).

In some embodiments, the purified peptide contains less than 0.25% acetate (eg, <0.2 or <0.1%).

In some embodiments, the purified peptide is substantially free of topoisomers (eg, <0.4, <0.3, <0.2, or <0.1%).

In some embodiments, the purified peptide is substantially free of iso-Asp2-plecanatide (RRT 0.96-0.97) (e.g., <0.4, <0.3, <0.2, or <0, 1%).

In some embodiments, the purified peptide has a chromatographic purity of at least 96%, at least 97%, or at least 98%. For example, a GCC agonist peptide has a chromatographic impurity content of 4% or less, 3.5% or less, 3% or less, 2.5% or less, 2% or less, 1.5% or less, or 1% or less. . The chromatographic content of impurities is determined as the total area percentage for impurities by HPLC. The chromatographic content of impurities includes

- 13 042294 content of topoisomers. Impurities do not include any pharmaceutically acceptable excipient used to formulate the drug.

In some embodiments, the purified peptide is substantially free of contaminants resulting from the peptide preparation process, such as organic solvents used in the process, e.g., ammonium, acetonitrile, acetamide, alcohol (e.g., methanol, ethanol, or isopropanol ), TFA, ether or other contaminants. In this context, substantially free from contaminants means that the content of contaminants of the peptide at the end of the purification process is preferably less than 0.5%, less than 0.3%, less than 0.25%, less than 0.1%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, less than 0.01%, less than 0.005%, less than 0.003%, or less than 0.001% of the total peptide weight. For example, the purified peptide contains <50 ppm acetamide (e.g., <35 or <18 ppm), <300 ppm acetonitrile (e.g., <250 ppm), <1000 ppm TFA (e.g., <400, <300, <200, <100 , or <50 ppm), <2000 ppm isopropanol (e.g. <1500, <1000, <500, <400, <300, <200, <100, <50 or <20 ppm) and/or <0.25% acetate (eg, <0.2 or <0.1%). The content of contaminants can be determined by conventional methods such as gas chromatography. Preferably, the amounts of residual solvents in the purified peptide of the invention are less than the limits set forth in ICH guidelines, for example, IMPURITIES: GUIDELINE FOR RESIDUAL SOLVENTS Q3C(R5) (available at http://www.ich.org/fileadmin/Public Web Site/ ICH Products/Guide lines/Quality/Q3C/Step4/Q3C_R5_Step4.pdf).

In some embodiments, the purified peptide contains less than 0.3% (eg, <0.15%) alpha-Asp-9-plenacanatide (RRT 1.33).

In some embodiments, the purified peptide has a bulk density of at most 0.09 g/ml, at most 0.08 g/ml, at most 0.07 g/ml, at most 0.06 g/ml, at most 0, 05 g/ml, not more than 0.04 g/ml or not more than 0.03 g/ml.

In some embodiments, the purified peptide is substantially free of iso-Asp2-plecanatide (RRT~0.96-0.97). In this context, essentially free from u3o-Asp2-plecanatide means that the content of iso-Asp2-plecanatide in the peptide at the end of the purification process is preferably less than 2%, less than 1.5%, less than 1.25%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0 1%, based on the total weight of the peptide.

In some embodiments, the purified peptide is substantially free of topoisomers. In this context, substantially free of topoisomers means that the topoisomer content of the peptide at the end of the purification process is preferably less than 2%, less than 1.5%, less than 1.25%, less than 1%, less than 0.9%, less than 0. 8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1%, of the total peptide weight .

In some embodiments, the purified peptide is substantially free of water. In this context, substantially free of water means that the water content of the peptide at the end of the purification process is preferably less than 10%, 9%, 8%, 7%, less than 6%, less than 5%, less than 4.5%, less than 4.25%. , less than 4%, less than 3.5%, less than 3%, less than 2.5%, less than 2%, less than 1.5%, less than 1%, less than 0.5%, less than 0.25%, or less than 0 1%, based on the total weight of the peptide.

For example, the purified peptide has a particle size distribution characterized by a D50 value of approximately 600 µm as measured by light scattering with an air dispersant. In comparison, peptides purified by the lyophilization and precipitation methods described in W2012/118972 have D50 values of approximately 180-250 μm and approximately 300 μm, respectively.

For example, a purified peptide produced by the methods of the invention has an acceptable size distribution for a pharmaceutical formulation. In one embodiment, the peptide (e.g., SP-304) has a size distribution (e.g., average size 80-120 µm) comparable to the size distribution of a pharmaceutical excipient (e.g., microcrystalline cellulose) used in a formulation, e.g., in a unit dosage form. form of 3 mg/day. The size distribution of the purified peptide may vary based on a single dose. For example, when a single dose is less than 3 mg/day, the purified peptide in the pharmaceutical formulation has a smaller average size than at a dose of 3 mg/day. For example, a purified peptide produced by the methods of the invention is milled to achieve an acceptable size distribution.

The size distribution of the peptide of the invention can be determined by conventional methods such as sieve analysis, shading or dynamic light scattering assays.

In one embodiment, the invention relates to a method for purifying peptides to be purified, including peptides synthesized by the hybrid methods described herein. The method includes, for example, the steps (denoted as isolation method 2) illustrated in Scheme 12 below as compared to the isolation steps described in WO2012/118972 (denoted as isolation method 1).

- 14 042294

Scheme 12.

In one embodiment, the purification method of the invention includes the following steps.

Monocyclization of linear crude peptide: First, a disulfide bond is formed between Cys4 and Cys12 SP-304 by H ₂ O ₂ oxidation at pH 8.0-8.5 to obtain a monocyclized crude peptide. The linear crude peptide is slowly added and dissolved in 0.5% ammonium acetate buffer in water pre-added with H ₂ O ₂ at a ratio of 100:9 grams of peptide to ml of H ₂ O ₂ to obtain a final concentration of the crude peptide of approximately 1.0 mg/ml . The pH of the solution is then adjusted to 8.5 with NH ₄ OH solution while stirring outdoors. The oxidative monocyclization reaction is monitored, for example, using HPLC method 2 as described in Example 8. When the area % for the linear crude peptide is <5.0% of the area % for the monocyclized peptide, the oxidation reaction is stopped by adjusting the pH of the peptide solution to 1.7 -2 using TFA solution. The peptide solution is then transferred to the next step to form a dicyclized peptide.

- 15 042294

Dicyclization of the monocyclized peptide.

A disulfide bond between Cys7 and Cys15 is formed with 3.0% (w/v) I2 in acetonitrile solution. The disulfide bridge is obtained while simultaneously removing the Acm side chain protecting groups on the remaining Cys residues. The oxidative dicyclization reaction is monitored, for example, using HPLC method 2. The excess iodine is neutralized with a solution of 0.1 M ascorbic acid in water. After completion of the reaction, the pH of the dicyclized peptide was adjusted to 6.57 using a solution of NH ₄ OH, and a material for primary purification was obtained.

Primary Purification of Dicyclized Crude Peptide: Then, the dicyclized crude peptide solution resulting from the oxidation steps is applied to a preparative RP-HPLC column packed with C18 reverse phase chromatography resin, controlled by a preparative HPLC system. The peptide is eluted in 1% TEAP in water, pH7/ACN buffer system. HPLC method 1 as described in example 8, for example, is used to clarify the percentage purity of fractions and pools obtained during the primary purification.

Purification of the recycled dicyclized peptide pool(s): After primary purification, fractions requiring further purification are purified based on the purity of the fraction pools using 1%TEAP in water, pH7/ACN or 0.2% acetic acid in water/ ACN. Then, the dicyclized crude peptide solution obtained from the oxidation steps is applied to a preparative RP-HPLC column packed with C18 reverse phase chromatography resin controlled by a preparative HPLC system. The peptide is eluted in 1% TEAP in water, pH7/ACN buffer system. HPLC method 1, as described in example 8, for example, is used to clarify the percentage purity of fractions and pools obtained during the re-purification.

Secondary purification and salt exchange: After secondary purification, fractions requiring further purification are purified based on the purity of fraction pools using 1% TEAP in water, pH7/ACN or 0.2% acetic acid in water/ACN buffer system. HPLC method 1, as described in example 8, for example, is used to clarify the percentage purity of fractions and pools obtained during the secondary purification. The SALC method 1 as described in Example 8, for example, is used to refine the percentage purity of the main pool obtained during the entire purification run before proceeding to the next step.

Solvent exchange: Material meeting the criteria of the main pool is loaded onto a preparative RP-HPLC column from the flanged end in the reverse direction, washed with a water/isopropanol solution (e.g. 99:1 water to isopropanol) in the reverse direction, and eluted with a water solution /isopropanol (for example, with a ratio of water to isopropanol 60:40) in the forward direction. The collected peptide solution is tested by HPLC method 1 to ensure purity, then the peptide solution is filtered, rotary evaporated to remove excess isopropanol (e.g., to less than 5%), followed by partial lyophilization, e.g., for at least 96 hours .

Dilution and Final Lyophilization: The partially lyophilized dry peptide is diluted in water to obtain a homogeneous batch. A buffer such as ammonium acetate (eg, 0.5% (w/w) of dry peptide) is added to the solution and mixed until the ammonium acetate and peptide are dissolved. The material can be analyzed by SELC method 1 to check the purity. The dissolved material can be placed in a lyophilizer tray and kept under vacuum, for example, for at least 120 hours to obtain the final dry peptide material.

1.1. GCC agonists.

GCC agonists produced by the methods of the invention can bind to guanylate cyclase C and stimulate intracellular production of cGMP. Optionally, GCC agonists induce apoptosis and inhibit epithelial cell proliferation. The term guanylate cyclase C refers to a transmembrane form of guanylate cyclase that acts as an intestinal receptor for thermostable toxin (ST) peptides secreted by enterobacteria. Guanylate cyclase C is also a receptor for the natural peptides guaniline and uroguaniline. It is not excluded that there may be different receptors for each of these peptides. Thus, the term guanylate cyclase C may also encompass the class of transmembrane guanylate cyclase receptors expressed on the epithelial cells lining the gastrointestinal mucosa.

The term GCC agonist refers to both peptides and non-peptide compounds, such as compounds that bind to intestinal guanylate cyclase C and stimulate intracellular production of cGMP. When a GCC agonist is a peptide, the term encompasses biologically active fragments of such peptides and propeptides that bind to guanylate cyclase C and stimulate intracellular production of cGMP.

1.1.1. GCC agonist peptides.

GCC agonists useful in the methods of the invention are preferably peptides. In some embodiments, the GCC agonist peptide is less than 30 amino acids in length. In specific embodiments, the GCC agonist peptide is less than or

- 16 042294 equal to 30, 25, 20, 15, 14, 13, 12, 11, 10 or 5 amino acids. Examples of GCC agonist peptides for use in the compositions and methods of the invention include those described in U.S. Patent Nos. 7,879,802 and 8,034,782 and U.S. Patent Publication Nos. US2010-0069306 and US2010-0120694, the contents of each of which are incorporated herein by reference.

Specific examples of GCC agonist peptides that can be purified or prepared by the methods of the invention include those described in Tables I-VII below. As used in Tables I-VII, the terms PEG3 or 3PEG refer to polyethylene glycol such as aminoethyloxyethyloxyacetic acid (AeeA) and its polymers. As used herein, the term AMIDE is intended to mean that a terminal carboxylic acid is substituted with an amide group, i.e. terminal COOH replaced by CONH ₂ .

The term _Xaa refers to any natural or non-natural amino acid or amino acid analogue. The term _Maa refers to cysteine (Cys), penicillamine (Pen), homocysteine or 3-mercaptoproline. The term Xaa _n1 is intended to refer to the amino acid sequence of any natural or non-natural amino acid or amino acid analog of one, two or three residues in length; Xaa _n2 is intended to denote an amino acid sequence of length zero or one residue; and Xaa _n3 is intended to represent an amino acid sequence of length zero, one, two, three, four, five, or six residues. In addition, any amino acid represented by Xaa, Xaani, Xaa _n2 or Xaa _n3 may be an L-amino acid, a D-amino acid, a methylated amino acid, or any combination thereof. Optionally, any GCC agonist peptide represented by Formulas I-XX in Tables may contain one or more polyethylene glycol residues at the N-terminus, C-terminus, or both.

In specific embodiments, the GCC agonist, purified or obtained by the methods of the invention, contains a peptide selected from SEQ ID NO: 1-251, the sequences of which are listed below in tables I-VII below. In one embodiment, the GCC agonist purified by the methods of the invention contains a peptide designated by SEQ ID NO: 1, 9, or 104.

In specific embodiments, the GCC agonist produced by the methods of the invention comprises a peptide that is substantially equivalent to a peptide selected from SEQ ID NO: 1-251. The term substantially equivalent refers to a peptide having an amino acid sequence equivalent to that of the binding domain, where specific residues can be deleted or replaced with other amino acids without impairing the ability of the peptide to bind to the gut guanylate cyclase receptor and promote fluid and electrolyte transport.

In a specific embodiment, the GCC agonist obtained by the methods of the invention contains peptides that are analogues of a peptide selected from SEQ ID NO: 1-251. In particular, these analogs contain α-aminoadipic acid (Aad), preferably at the 3rd position from the N-terminus of each peptide, or at the N-terminus side adjacent to the first cysteine residue (Cys). 251.

In specific embodiments, the GCC agonist peptides are analogues of uroguaniline or ST bacterial peptide. Uroguaniline is a circulating peptide hormone with natriuretic activity. The ST peptide is a member of a family of thermostable enterotoxins (ST peptides) secreted by pathogenic strains of E. coli and other enterobacteria that activate the guanylate cyclase receptor and cause secretory diarrhea. Unlike bacterial ST peptides, the binding of uroguaniline to the guanylate cyclase receptor is dependent on physiological pH in the gut. Therefore, uroguaniline would be expected to regulate fluid and electrolyte transport in a pH dependent manner without causing severe diarrhea.

The GCC agonist peptides prepared by the methods of the invention may be polymers of L-amino acids, D-amino acids, or a combination of both. For example, in various embodiments, the peptides are D-retro-inverso peptides. The term retro inverse isomer refers to an isomer of a linear peptide in which the direction of the sequence is reversed and the chirality of each amino acid residue is inverted. See, for example, Jameson et al., Nature, 368, 744746 (1994); Brady et al., Nature, 368, 692-693 (1994). The end result of the combination of D-enantiomers and reverse synthesis is that the positions of the carbonyl and amino groups in each amide bond are reversed, while the position of the side groups at each alpha carbon is retained. Unless specifically stated otherwise, it is contemplated that any given L-amino acid sequence of the invention can be converted to a D-retro-inverso peptide by synthesizing the reverse sequence of the corresponding native L-amino acid sequence.

GCC agonist peptides prepared by the methods of the invention are capable of inducing intracellular cGMP production in cells and tissues expressing guanylate cyclase C. In certain embodiments, the GCC agonist peptide stimulates 5, 10, 20, 30, 40, 50, 75, 90 % or more intracellular cGMP compared to natural GCC agonists such as uroguaniline, guaniline, or ST peptides. Optionally, the GCC agonist peptide stimulates 5%, 10%, 20%, 30%, 40%, 50%, 75%, 90% or more of intracellular cGMP compared to SP-304 (SEQ ID NO: 1). In further embodiments, the GCC agonist peptide stimulates apoptosis, such as programmed cell death, or activates the cystic fibrotic transmembrane conductance regulator (CFTR).

- 17 042294

In some embodiments, the GCC agonist peptides produced by the methods of the invention are more stable than natural GCC agonists and/or SP-304 (SEQ ID NO: 1), SP339 (linaclotide) (SEQ ID NO: 55), or SP -340 (SEQ ID NO: 56). For example, a GCC agonist peptide is degraded by 2, 3, 5, 10, 15, 20, 30, 40, 50, 75, 90% or less compared to natural GCC agonists and/or SP-304, SP-339 (linaclotide ) or SP-340. In specific embodiments, GCC agonist peptides for use in the compositions and methods of the invention are more stable with respect to proteolytic cleavage than natural GCC agonists and/or SP-304 (SEQ ID NO: 1), SP-339 (linaclotide) (SEQ ID NO: 55) or SP-340 (SEQ ID NO: 56). In one embodiment, the GCC agonist peptide is pegylated to render the peptides more resistant to proteolysis by gastrointestinal enzymes. In a preferred embodiment, the GCC agonist peptide is pegylated with an aminoethyloxyethyloxyacetic acid (Aeea) moiety at its C-terminus, at its N-terminus, or at both ends.

Specific examples of GCC agonist peptides that can be produced by the methods of the invention include peptides selected from the group indicated by SEQ ID NOs: 1-251.

In one embodiment, the GCC agonist peptide is a peptide having an amino acid sequence from any of Formulas X-XVII (eg, SEQ ID NOs: 87-98).

In some embodiments, GCC agonist peptides include peptides having the formula I amino acid sequence, wherein at least one formula I amino acid is a D-amino acid or a methylated amino acid and/or the amino acid at position 16 is a series. Preferably, the amino acid at position 16 of formula I is a D-amino acid or a methylated amino acid. For example, the amino acid at position 16 of formula I is d-leucine or d-serine. Optionally, one or more of the amino acids at positions 1-3 of formula I are D-amino acids or methylated amino acids, or a combination of D-amino acids or methylated amino acids. For example, Asn ¹ , Asp ² or Glu ³ (or a combination thereof) of Formula I is a D-amino acid or a methylated amino acid. Preferably, the amino acid at position Xaa ⁶ of formula I is leucine, serine or tyrosine.

In alternative embodiments, GCC agonist peptides include peptides having an amino acid sequence of formula II, wherein at least one amino acid of formula II is a D-amino acid or a methylated amino acid. Preferably, the amino acid designated Xaa _n2 of Formula II is a D-amino acid or a methylated amino acid. In some embodiments, the amino acid designated Xaa _n2 of Formula II is leucine, d-leucine, serine, or d-serine. Preferably, one or more of the amino acids designated Xaa _n1 of formula II is a D-amino acid or a methylated amino acid. Preferably, the amino acid at position Xaa ⁶ of formula II is leucine, serine or tyrosine.

In some embodiments, GCC agonist peptides include peptides having the formula III amino acid sequence, wherein at least one formula III amino acid is a D-amino acid or a methylated amino acid and/or Maa is not a cysteine. Preferably, the amino acid designated Xaa _n2 of formula III is a D-amino acid or a methylated amino acid. In some embodiments, the amino acid designated Xaa _n2 of Formula III is leucine, d-leucine, serine, or d-serine. Preferably, one or more of the amino acids designated Xaan1 of formula III is a D-amino acid or a methylated amino acid.

Preferably, the amino acid at position Xaa6 of formula III is leucine, serine or tyrosine.

In other embodiments, GCC agonist peptides include peptides having the formula IV amino acid sequence, wherein at least one formula IV amino acid is a D-amino acid or a methylated amino acid and/or Maa is not a cysteine. Preferably, Xaa _n2 of formula IV is a D-amino acid or a methylated amino acid. In some embodiments, the amino acid designated Xaa _n2 of Formula IV is leucine, d-leucine, serine, or d-serine. Preferably, one or more of the amino acids designated Xaa _n1 of formula IV is a D-amino acid or a methylated amino acid. Preferably, the amino acid designated Xaa ⁶ of formula IV is leucine, serine or tyrosine.

In further embodiments, GCC agonist peptides include peptides having the formula V amino acid sequence, wherein at least one formula V amino acid is a D-amino acid or a methylated amino acid. Preferably, the amino acid at position 16 of Formula V is a D-amino acid or a methylated amino acid. For example, the amino acid at position 16 (ie Xaa ¹⁶ ) of Formula V is d-leucine or d-serine. Optionally, one or more amino acids at position 1-3 of formula V are D-amino acids or methylated amino acids, or a combination of D-amino acids

- 18 042294 or methylated amino acids. For example, Asn ¹ , Asp ² or Glu ³ (or a combination thereof) of Formula V are D-amino acids or methylated amino acids. Preferably, the amino acid designated Xaa ⁶ of formula V is leucine, serine or tyrosine.

In additional embodiments, the GCC agonist peptides include peptides having the amino acid sequence of Formula VI, VII, VIII, or IX. Preferably, the amino acid at position 6 of formula VI, VII, VIII or IX is leucine, serine or tyrosine. In some aspects, the amino acid at position 16 of formula VI, VII, VIII, or IX is leucine or serine. Preferably, the amino acid at position 16 of Formula V is a D-amino acid or a methylated amino acid.

In additional embodiments, the GCC agonist peptides include peptides having the amino acid sequence of Formula X, XI, XII, XIII, XIV, XV, XVI, or XVII. Optionally, one or more amino acids of formulas X, XI, XII, XIII, XIV, XV, XVI or XVII are D-amino acids or methylated amino acids. Preferably, the amino acid at the carboxy-terminus of the peptides of formulas X, XI, XII, XIII, XIV, XV, XVI or XVII is a D-amino acid or a methylated amino acid. For example, the amino acid at the carboxy-terminus of the peptides of formulas X, XI, XII, XIII, XIV, XV, XVI, or XVII is D-tyrosine.

Preferably, the amino acid designated Xa ⁶ of formula XIV is tyrosine, phenylalanine, or serine. Most preferably, the amino acid designated Xaa ⁶ of Formula XIV is phenylalanine or serine. Preferably, the amino acid designated Xaa ⁴ of formula XV, XVI or XVII is tyrosine, phenylalanine or serine. Most preferably, the amino acid at position Xaa ⁴ of formula V, XVI or XVII is phenylalanine or serine.

In some embodiments, the GCRA peptides include peptides containing the amino acid sequence of Formula XVIII. Preferably, the amino acid at position 1 of formula XVIII is glutamic acid, aspartic acid, glutamine or lysine. Preferably, the amino acid at positions 2 and 3 of formula XVIII is glutamic acid or aspartic acid. Preferably, the amino acid at position 5 is glutamic acid. Preferably, the amino acid at position 6 of formula XVIII is isoleucine, valine, serine, threonine, or tyrosine. Preferably, the amino acid at position 8 of formula XVIII is valine or isoleucine. Preferably, the amino acid at position 9 of formula XVIII is asparagine. Preferably, the amino acid at position 10 of formula XVIII is valine or methionine. Preferably, the amino acid at position 11 of formula XVIII is alanine. Preferably, the amino acid at position 13 of Formula XVIII is threonine. Preferably, the amino acid at position 14 of Formula XVIII is glycine. Preferably, the amino acid at position 16 of formula XVIII is leucine, serine or threonine.

In alternative embodiments, the GCRA peptides include peptides containing the amino acid sequence of formula XIX. Preferably, the amino acid at position 1 of formula XIX is the series or asparagine. Preferably, the amino acid at position 2 of formula XIX is histidine or aspartic acid. Preferably, the amino acid at position 3 of formula XIX is threonine or glutamic acid. Preferably, the amino acid at position 5 of Formula XIX is glutamic acid. Preferably, the amino acid at position 6 of formula XIX is isoleucine, leucine, valine or tyrosine. Preferably, the amino acid at position 8, 10, 11 or 13 of formula XIX is alanine. Preferably, the amino acid at position 9 of formula XIX is asparagine or phenylalanine. Preferably, the amino acid at position 14 of formula XIX is glycine.

In further embodiments, the GCRA peptides include peptides containing the amino acid sequence of Formula XX. Preferably, the amino acid at position 1 of formula XX is glutamine. Preferably, the amino acid at position 2 or 3 of formula XX is glutamic acid or aspartic acid. Preferably, the amino acid at position 5 of formula XX is glutamic acid. Preferably, the amino acid at position 6 of formula XX is threonine, glutamine, tyrosine, isoleucine or leucine. Preferably, the amino acid at position 8 of formula XX is isoleucine or valine. Preferably, the amino acid at position 9 of formula XX is asparagine. Preferably, the amino acid at position 10 of formula XX is methionine or valine. Preferably, the amino acid at position 11 of formula XX is alanine. Preferably, the amino acid at position 13 of formula XX is threonine. Preferably, the amino acid at position 1 of formula XX is glycine. Preferably, the amino acid at position 15 of formula XX is tyrosine. Optionally, the amino acid at position 15 of formula XX is two amino acids long and is cysteine (Cys), penicillamine (Pen), homocysteine or 3-mercaptoproline and serine, leucine or threonine.

In certain embodiments, one or more amino acids of the agonist peptides

- 19 042294

GCC is replaced by a non-natural amino acid or a natural or non-natural amino acid analog. Such amino acids and amino acid analogs are known in the art. See, for example, Hunt, The NonProtein Amino Acids, in Chemistry and Biochemistry of the Amino Acids, Barrett, Chapman, and Hall, 1985. In some embodiments, the amino acid is replaced with a naturally occurring, non-essential amino acid, such as taurine. Non-limiting examples of natural amino acids that can be replaced with non-protein amino acids include the following: (1) an aromatic amino acid can be replaced by 3,4-dihydroxy-L-phenylαlanine, 3-iodo-L-tyrosine, triiodothyronine, L-thyroxine, phenylglycine (Phg ) or nortyrosine (norTyr); (2) Phg and norTyr, and other amino acids, including Phe and Tyr, can be substituted with, for example, halogen, -CH ₃ , -OH, -CH ₂ NH ₃ , -C(O)H, -CH ₂ CH ₃ , - CN, -CH ₂ CH ₂ CH ₃ , -SH or another group; (3) glutamine residues can be replaced with gamma-hydroxy-Glu or gamma-carboxy-Glu; (4) tyrosine residues can be replaced with an alpha-substituted amino acid such as L-alpha-methylphenylalanine or analogues such as: 3-amino-Tyr; Tyr (CH ₃ ); Tyr (PO ₃ (CH ₃ ) ₂ ); Tyr(SO ₃ H); beta-cyclohexyl-Ala; beta-(1cyclopentenyl)-Ala; beta-cyclopentyl-Ala; beta-cyclopropyl-Ala; beta-quinolyl-Ala; beta-(2-thiazolyl)Ala; beta-(triazol-1-yl)-Ala; beta-(2-pyridyl)-Ala; beta-(3-pyridyl)-Ala; amino Phe; fluoro-Phe; cyclohexyl-Gly; tBu-Gly; beta-(3-benzothienyl)-Ala; beta-(2-thienyl)-Ala; 5-methyl-Trp; and A-methyl-Trp; (5) proline residues can be replaced with homopro(L-pipecolic acid); hydroxy-Pro; 3,4-dehydro-Pro; 4-Fluoro-Pro; or alpha-methyl-Pro, or N(αα)-C(alpha)-cyclized amino acid analogs with the structure: n=0, 1, 2, 3; and (6) alanine residues can be replaced with an alpha-substituted or N-methylated amino acid such as alpha-aminoisobutyric acid (aib), L/D-alpha-ethylalanine (L/D-isovaline), L/D-methylvaline, or L /D-alpha-methylleucine or a non-natural amino acid such as beta-fluoro-Ala. Alanya can also be replaced by: n=0, 1, 2, 3 residues. Glycine can be replaced with alpha-aminoisobutyric acid (aib) or L/D-alpha-ethylalanine (L/D-isovalin).

Additional examples of non-natural amino acids include: a non-natural tyrosine analog; unnatural analogue of glutamine; unnatural analogue of phenylalanine; non-natural analogue of serine; unnatural analogue of threonine; amino acid substituted by alkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynyl, ether, thiol, sulfonyl, selenium, ester, thioacid, borate, boronate, phospho, phosphono, phosphine, heterocycle , enone, imine, aldehyde, hydroxylamine, keto or amino, or any combination thereof; an amino acid with a photoactivatable crosslinking group; spin-labeled amino acid; a fluorescent amino acid; an amino acid with a new functional group; an amino acid that interacts covalently or non-covalently with another molecule; a metal-binding amino acid; an amino acid amidated at a site that is not naturally amidated, a metal-containing amino acid; radioactive amino acid; a photoscreening and/or photoisomerizable amino acid; an amino acid containing biotin or a biotin analog; a glycosylated or carbohydrate-modified amino acid; a ketogenic amino acid; an amino acid containing polyethylene glycol or polyether; a heavy atom substituted amino acid (eg, containing deuterium, tritium, 13C, 15N, or 180 amino acids); a chemically cleavable or photodegradable amino acid; an amino acid with an extended side chain; an amino acid containing a toxic group; a sugar-substituted amino acid, for example, a sugar-substituted series or the like; an amino acid containing a sugar attached to a carbon atom; an amino acid with a redox potential; an acid containing an α-hydroxyl group; an amino acid containing an aminothioacid; α,α-disubstituted amino acid; β-amino acid; a cyclic amino acid other than proline; O-methyl-b-tyrosine; L-3-(2-naphthyl)alanine; 3-methyl-phenylalanine; ρacetyl-b-phenylalanine; O-4-allyl-L-tyrosine; 4-propyl-L-tyrosine; tri-O-acetyl-GlcNAc β-serine; Ldopa; fluorinated phenylalanine; isopropyl-b-phenylalanine; p-azido-b-phenylalanine; p-acyl-L-phenylalanine; p-benzoyl-L-phenylalanine; L-phosphoserine; phosphonoserine; phosphonothyrosine; piodophenylalanine; 4-fluorophenylglycine; p-bromophenylalanine; p-amino-G-phenylalanine; isopropyl-L-phenylalanine; L-3-(2-naphthyl)alanine; D-3-(2-naphthyl)alanine (dNal); amino-, isopropyl-, or O-allyl-containing analogue of phenylalanine; dopa, O-methyl-L-tyrosine; glycosylated amino acid; p(propargyloxy)phenylalanine; dimethyllysine; hydroxyproline; mercaptopropionic acid; methyllysine; 3-nitrotyrosine; norleucine; pyroglutamic acid; Z (carbobenoxyl); ε-acetyl lysine; β-alanine; an aminobenzoyl derivative; aminobutyric acid (Abu); citrulline; aminohexanoic acid; aminoisobutyric acid (AIB); cyclohexylalanine; d-cyclohexylalanine; hydroxyproline; nitroarginine; nitrophenylalanine; nitrotyrosine; norvaline; octahydroindole carboxylate; ornithine (Orn); penicillamine (PEN); tetrahydroisoquinoline; acetamidomethyl-protected amino acids; and pegylated amino acids. Additional examples of non-natural amino acids and amino acid analogues can be found in US20030108885, US20030082575, Us2o06OO19347 (paragraphs 410-418) and the references cited therein. The polypeptides of the invention may include further modifications, including those described in US20060019347, paragraph 589. Exemplary GCC agonist peptides containing unnatural amino acids include, for example, SP-368 and SP-369.

In some embodiments, the GCC agonist peptides are cyclic

- 20 042294 peptides. Cyclic GCC agonist peptides can be prepared by methods known in the art. For example, macrocyclization is often carried out by forming an amide bond between the N- and C-termini of the peptide, between the side chain and the N- or C-terminus [for example, with K ₃ Fe(CN) ₆ at pH 8.5] (Samson et al., Endocrinology, 137: 5182-5185 (1996)), or between the side chains of two amino acids such as cysteine. See, for example, DeGrado, Adv Protein Chem, 39: 51-124 (1988). In various embodiments, the GCC agonist peptides are [4,12; 7.15] bicycles.

In specific embodiments, one or both of the Cys residues that normally form a disulfide bond in the GCC agonist peptide are replaced with homocysteine, penicillamine, 3-mercaptoproline (Kolodziej et al. 1996 Int. J. Pept. Protein Res. 48:274), β,β-dimethylcysteine (Hunt et al. 1993 Int. J. Pept. Protein Res. 42:249) or diaminopropionic acid (Smith et al. 1978 J. Med. Chem. 2 1:117) to form alternative internal crosslinks in positions of conventional disulfide bonds.

In specific embodiments, _one or more _disulfide bonds in the _GCC peptide agonist are replaced with alternative covalent cross-links, e.g. bond, lactam bridge, carbamoyl bond, urea bond, thiourea bond, phosphonate ester bond, alkyl bond (-CH2CH2CH2CH2-), alkenyl bond (-CH ₂ CH=CHCH ₂ -), ether bond (-CH ₂ CH ₂ OCH ₂ - or -CH ₂ OCH ₂ CH ₂ -), a thioether bond (-CH2CH2SCH2- or -CH2SCH2CH2-), an amine bond (-CH2CH2NHCH2- or -CH2NHCH2CH2-) or a thioamide bond (-CH2C (S)NHCH ₂ - or -CH2NHC (S) CH2-). For example, Ledu et al. (Proc. Natl. Acad. Sci. 100:11263-78, 2003) describes methods for preparing lactam and amide crosslinks. Exemplary GCC agonist peptides containing a lactam bridge include, for example, SP-370.

In specific embodiments, the GCC agonist peptides have one or more conventional polypeptide linkages replaced with alternative linkages. Such substitutions may increase the stability of the polypeptide. For example, replacing a polypeptide bond between an amino-terminal residue and an aromatic residue (eg, Tyr, Phe, Trp) with an alternative bond can reduce cleavage by carboxypeptidases and increase gastrointestinal half-life. Bonds that can be substituted for polypeptide bonds include: retro-inverse bond (C(O)-NH instead of NH-C(O); reduced amide bond (NH-CH2); thiomethylene bond (S-CH2 or CH2-S ); an oxomethylene bond (O-CH2 or _CH2 -O); an ethylene bond (CH2-CH2); a thioamide bond (C(S)-NH); a trans-olefin bond (CH=CH); a fluorine-substituted trans-olefin bond (CF=CH); a ketomethylene bond (C(O)-CHR or CHR-C(O) where R is H or CH ₃ ; and a fluoro-ketomethylene bond (C(O)-CFR or CFR-C(O ), where R represents H or F, or CH ₃ .

In specific embodiments, the GCC agonist peptides are modified using conventional modifications. Modifications can be made at the amino (N-), carboxy (C-) termini, within or in combination of any of the above. In one of the aspects described herein, more than one type of polypeptide modification may be present. Modifications include, but are not limited to: acetylation, amidation, biotinylation, cinnamoylation, farnesylation, formylation, myristoylation, palmitoylation, phosphorylation (Ser, Tyr, or Thr), stearoylation, succinylation, sulfurylation, and cyclization (via disulfide bridges or amide cyclization), and modification by Cys3 or Cys5. The GCC agonist peptides described herein can also be modified with 2,4-dinitrophenyl (DNP), DNP-lysine, modified with 7-amino-4-methylcoumarin (AMC), fluorescein, NBD (7-nitrobenz-2-oxa- 1,3-diazole), p-nitroanilide, rhodamine B, EDANS (5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid), dabcyl, dabsyl, dansyl, Texas red, FMOC, and Tamra (tetramethylrhodamine). The GCC agonist peptides described herein can also be conjugated to, for example, polyethylene glycol (PEG); alkyl groups (eg C1- _C20 linear or branched alkyl groups); fatty acid groups; combinations of PEG, alkyl groups and fatty acid groups (see US Pat. No. 6,309,633; Soltero et al., 2001 Innovations in Pharmaceutical Technology 106-110); BSA and KLH (saucer hemocyanin). The addition of PEG and other polymers that can be used to modify the polypeptides of the invention is described in US20060 19347 Section IX.

The GCC agonist peptide may also be a derivative of the GCC agonist peptide described herein. For example, the derivative includes hybrid and modified forms of GCC agonist peptides in which specific amino acids are deleted or substituted. The modification may also include glycosylation. Preferably, when the modification is an amino acid substitution, it is a conservative substitution at one or more positions predicted to be amino acid residues not necessary for the biological activity of the peptide. A conservative substitution is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids with basic side chains.

- 21 042294 chains (eg lysine, arginine, histidine), acidic side chains (eg aspartic acid, glutamic acid), uncharged polar side chains (eg glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (eg, threonine, valine, isoleucine), and aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine ).

In one embodiment, the GCC agonist peptide produced by the methods described herein is subjected to random mutagenesis to identify mutants with biological activity.

In one embodiment, the methods of the invention can be used to produce a GCC agonist peptide that is substantially homologous to the GCC agonist peptide described herein. Such substantially homologous peptides can be isolated by cross-reaction with antibodies against the GCC agonist peptide described herein.

Additional examples of GCC agonist peptides that can be produced by the methods of the invention can be found in Tables I-VII below.

1.1.2. Alternative methods for the production of GCC agonist peptides and fragments thereof.

GCC agonist peptides and fragments thereof can be prepared using art-recognized methods such as molecular cloning, peptide synthesis, or site-directed mutagenesis.

In addition to the conventional solution or solid phase peptide synthesis described above, GCC agonist peptides or fragments thereof can be obtained by modern cloning techniques. For example, GCC agonist peptides are produced either in bacteria, including but not limited to E. coli, or in other currently available systems for the production of polypeptides or proteins (for example, Bacillus subtilis, baculovirus expression systems using Drosophila Sf9 cells , expressing systems of yeast or filamentous fungi, expressing systems of mammalian cells), or they can be synthesized by chemical methods. If the GCC agonist peptide or peptide variant is intended to be produced in bacteria, eg E. coli, the nucleic acid molecule encoding the polypeptide may also encode a leader sequence allowing secretion of the mature polypeptide from the cell. Thus, a polypeptide coding sequence may include a pre-sequence and a pro-sequence of, for example, a naturally occurring bacterial ST polypeptide. The secreted mature polypeptide can be purified from the culture medium.

The sequence encoding the GCC agonist peptide described herein can be inserted into a vector capable of delivering and maintaining a nucleic acid molecule in a bacterial cell. The DNA molecule can be inserted into an autonomously replicating vector (suitable vectors include, for example, pGEM3Z and pcDNA3 and their derivatives). The vector nucleic acid may be bacterial or bacteriophage DNA such as bacteriophage lambda or M13 and derivatives thereof. Construction of a vector containing the nucleic acid described herein may be followed by transformation of a host cell, such as a bacterium. Suitable bacterial hosts include, but are not limited to, E. coli, B. subtilis, Pseudomonas, Salmonella. The genetic construct also includes, in addition to the coding nucleic acid molecule, elements allowing expression such as promoter and regulatory sequences. Expression vectors may contain transcription control sequences that control the initiation of transcription, such as promoter, enhancer, operator, and repressor sequences.

A variety of transcription control sequences are well known to those skilled in the art. The expression vector may also include a sequence for regulating translation (eg, a 5' untranslated sequence, a 3' untranslated sequence, or an internal ribosome binding site). The vector may be capable of autonomous replication, or it may integrate into the host's DNA to ensure stable production of the polypeptide.

A protein coding sequence comprising the GCC agonist peptide described herein can also be fused to a nucleic acid encoding a polypeptide affinity tag, e.g., glutathione S-transferase (GST), maltose binding protein E, protein A, FLAG tag , hexahistidine, myc label, or influenza virus HA label, to facilitate cleaning. A fusion with an affinity tag or reporter connects the reading frame of the polypeptide of interest to the reading frame of the gene encoding the affinity tag, such that a translational fusion is obtained. Expression of the fusion gene results in translation of a single polypeptide, including both the polypeptide of interest and the affinity tag. In some instances where affinity tags are used, a DNA sequence encoding a protease recognition site is inserted for fusion between the reading frames for the affinity tag and the polypeptide of interest.

- 22 042294

Genetic constructs and methods suitable for the production of immature and mature forms of GCC agonist peptides and variants described herein in protein-expressing systems other than bacteria, and well known to those skilled in the art, can also be used to produce polypeptides in biological system.

The peptides described herein can be modified by adding a second molecule that gives the peptide the desired property, such as increased half-life in the body, such as pegylation. Such modifications are also within the scope of the term variant as used herein.

Table I. GCRA peptides (SP-304 and derivatives)

Name- ing Position of disulfide bonds Structure SEQ ID NO SP-304 C4:C12, C7:C15 Asn ⁱ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys ^Val^AsrC-Val^-Ala^-Gys^-Thr^-Gly ¹⁴ Cys ¹⁵ -Leu ¹⁶ 1 SP-326 SZ:S11, S6:S14 Asp^GW-Cys^GW-LeiC-Cys^VaP-AsrA-Val^Ala^-Cys^-Thr^-Gly^-Cys ¹⁴ Leu ¹⁵ 2 SP-327 C2:C10, C5:C13 Asp ¹ -Glu ² -Cys ³ -Glu ⁴ -Leu ⁵ -Cys ⁶ -Val ⁷ -Asn ⁸ -Val ⁹ -Ala ¹⁰ -Cys ¹¹ -Thr ¹² -Gly ¹³ -Cys ¹⁴ 3 SP-328 C2:C10, C5:C13 Glu ¹ -Cys ² -Glu ³ -Leu ⁴ -Cys ⁵ -Val ⁶ -Asn ⁷ -Val ⁸ -Ala ⁹ -Cys ¹⁰ -Thr ¹¹ -Gly ¹² -Cys ¹³ -Leu ¹⁴ 4 SP-329 C2:C10, C5:C13 Glu ¹ -Cys ² -Glu ³ -Leu ⁴ -Cys ⁵ -Val ⁶ -Asn ⁷ -Val ⁸ -Ala ⁹ -Cys ¹⁰ -Thr ¹¹ -Gly ¹² -Cys ¹³ 5 SP-330 C1:C9, C4:C12 Cys ¹ -Glu ² -Leu ³ -Cys ⁴ -Val ⁵ -Asn ⁶ -Val ⁷ -Ala ⁸ -Cys ⁹ -Thr ¹⁰ -Gly ¹¹ -Cys ¹² -Leu ¹³ 6 SP-331 C1:C9, C4:C12 Cys ¹ -Glu ² -Leu ³ -Cys ⁴ -Val ⁵ -Asn ⁶ -Val ⁷ -Ala ⁸ -Cys ⁹ -Thr ¹⁰ -Gly ¹¹ -Cys ¹² 7 SP332 C4:C12, C7:C15 Asn ⁱ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys ^/ -Val ⁸ -Asn ⁹ -Val ^iU -Ala ⁱⁱ -Cys ⁱ² -Thr ⁱ³ -Gly ⁱ⁴ Cys ¹⁵ -dLeu ¹⁶ 8 SP-333 C4:C12,C7:C15 dAsn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys ^/ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ 9 SP-334 C4:C12, C7:C15 dAsn ⁱ -dAsp ² -Glu ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys ^/ -Val ⁸ -Asn ⁹ -Val ^iU -Ala ⁱⁱ -Cys ⁱ² -Thr ⁱ³ -Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ 10 SP-335 C4:C12,C7:C15 dAsn ⁱ -dAsp ² -dGlu ³ -Cys ⁴ -Glu ^i> -Leu ⁶ -Cys ^/ -Val ⁸ -Asn ⁹ -Val ^iU -Ala ⁱⁱ -Cys ⁱ² -Thr ⁱ³ Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ eleven SP-336 C4:C12, C7:C15 dAsn ⁱ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys ^/ -Val ⁸ -Asn ⁹ -Val ^iU -Ala ⁱⁱ -Cys ⁱ² -Thr ⁱ³ Gly ¹⁴ -Cys ¹⁵ -Leu ¹⁶ 12 SP-337 C4:C12, C7:C15 dAsn ⁱ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -dLeu ⁶ -Cys ^/ -Val ⁸ -Asn ⁹ -Val ^iU -Ala ⁱⁱ -Cys ⁱ² -Thr ⁱ³ -Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ 13 SP-338 C4:C12, C7:C15 Asn ⁱ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys ^/ -Val ⁸ -Asn ⁹ -Val ^iU -Ala ⁱⁱ -Cys ⁱ² -Thr ⁱ³ -Gly ⁱ⁴ Cys ¹⁵ 14 SP-342 C4:C12, C7:C15 PEG3-Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys ^/ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ - PEG3 15 SP-343 C4:C12, C7:C15 PEG3-dAsn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys ^/ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ - PEG3 16 SP-344 C4:C12, C7:C15 PEG3-dAsn ¹ -dAsp 2 -Glu ³ -Cys ^{4 -Glu b} -Leu ⁶ -Cys ^/ -Val ⁸ -Asn ⁹ -Val ^lu -Ala ¹¹ -Cys ¹² - Thr ¹³ -Gly ¹⁴ ~Cys ¹⁵ ~dLeu ¹⁶ - PEG3 17 SP-347 C4:C12, C7:C15 dAsn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys ^/ -Val ⁸ -Asn ⁹ -Val ^lu -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ -PEG3 18 SP-348 C4:C12, C7:C15 PEG3-Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys ^/ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ 19

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SP-350 C4:C12, 07:015 PEG3-dAsn ⁱ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys ^/ -Val ⁸ -Asn ⁹ -Val ^iU -Ala ⁱⁱ -Cys ⁱ² -Thr ⁱ³ -Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ 20 SP-352 04:012, 07:015 Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ^;> -Leu ^b -Cys '-VaY-Asny-Val^-Ala^-Cys^-Thr^-Gly ¹⁴ - Cys ¹⁵ -dLeu ¹⁶ -PEG3 21 SP-358 C4:C12,C7:C15 PEG3-dAsn ⁱ -dAsp ² -dGlu ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys ^/ -Val ⁸ -Asn ⁹ -Val ^iU -Ala ⁱⁱ -Cys ⁱ² Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ ~PEG3 22 SP-359 04:012,07:015 PEG3-dAsn ⁱ -dAsp ² -dGlu ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys ^/ -Val ⁸ -Asny-Val ^iU -Ala ⁱⁱ -Cys ⁱ² Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ 23 SP-360 04:012, 07:015 dAsn ⁱ -dAsp ² -dGlu ³ -Cys ⁴ -Glu ^b -Leu ⁶ -0ys ^/ -Val ⁸ -Asn ⁹ -Val ^iU -Ala ⁱⁱ -Cys ⁱ² -Thr ⁱ³ Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ ~PEG3 24 SP-361 04:012, 07:C15 dAsn ⁱ -dAsp ² -Glu ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys ^/ -Val ⁸ -Asn ^y -Val ^iU -Ala ⁱⁱ -Cys ⁱ² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ -PEG3 25 SP-362 04:012, 07:015 PEG3-dAsn ⁱ -dAsp ² -Glu ³ -Cys ⁴ -Glu ^b -Leu ⁶ -0ys '-YaT-AznY-YaA-AEa^-Cus ¹² Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ 26 SP-368 04:012, 07:C15 dAsn ⁱ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys ^/ -Val ³ -Asn ^y -Val ^iU -Ala ⁱⁱ -Cys ⁱ² -Thr ⁱ³ Gly ¹⁴ -Cys ¹⁵ -dNal ¹⁶ 27 SP-369 04:012, 07:015 dAsn ⁱ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys ^/ -AIB ⁸ -Asn ⁹ -AIB ^iU -Ala ⁱⁱ -Cys ⁱ² -Thr ⁱ³ Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ 28 SP-370 04:C12, 07:015 ^{dAsn 1} -Asp ^{2 -Glu 3 -Cys 4} -Glu ^b -Leu ^fc -Asp ^[ Lactam ^] 29 SP-371 04:012,07:015 dAsn ⁱ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -Tyr ⁶ -Cys ^/ -Val ³ -Asn ^y -Val ^iU -Ala ⁱⁱ -Cys ⁱ² -Thr ⁱ³ Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ thirty

SP-372 C4:C12,C7:C15 dAsn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -Ser ⁶ -Cys ^/ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ^{12 -Thr 13} -Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ 31 N1 C4:C12,C7:C15 PEG3-dAsn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -Tyr ⁶ -Cys ^/ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ - PEG3 32 N2 C4:C12,C7:C15 PEG3-dAsn ⁱ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -Tyr ⁶ -Cys ^/ -Val ⁸ -Asn ⁹ -Val ^iU -Ala ⁱⁱ -Cys ⁱ² -Thr ¹³ Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ 33 N3 C4:C12,C7:C15 dAsn ⁱ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -Tyr ⁶ -Cys ^/ -Val ⁸ -Asn ⁹ -Val ^iU -Ala ⁱⁱ -Cys ⁱ² -Thr ⁱ³ -Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ PEG3 34 N4 C4:C12,C7:C15 PEG3-dAsn ⁱ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -Ser ⁶ -Cys ^/ -Val ⁸ -Asn ⁹ -Val ^iU -Ala ⁱⁱ -Cys ⁱ² -Thr ¹³ Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ -PEG3 35 N5 C4:C12,C7:C15 PEG3-dAsn ⁱ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -Ser ⁶ -Cys ^/ -Val ⁸ -Asn ⁹ -Val ^iU -Ala ⁱⁱ -Cys ⁱ² -Thr ¹³ Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ 36 N6 C4:C12,C7:C15 dAsn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -Ser ⁶ -Cys ^/ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ -PEG3 37 N7 C4:C12,C7:C15 Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys '-VaY-AsY-Val^-Ala^-Cys^-Thr^-Gly ¹⁴ Cys ¹⁵ -Ser ¹⁶ 38 N8 C4:C12,C7:C15 PEG3-Asn ⁱ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys ^/ -Val ⁸ -Asn ⁹ -Val ^iU -Ala ⁱⁱ -Cys ⁱ² -Thr ⁱ³ Gly ¹⁴ -Cys ¹⁵ -Ser ¹⁶ -PEG3 39 N9 C4:C12,C7:C15 PEG3-Asn ⁱ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys ^/ -Val ⁸ -Asn ⁹ -Val ^iU -Ala ⁱⁱ -Cys ⁱ² -Thr ⁱ³ Gly ¹⁴ -Cys ¹⁵ -Ser ¹⁶ 40 N10 C4:C12,C7:C15 Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys '-VaY-AsY-Val^-Ala^-Cys^-Thr^-Gly ¹⁴ - Cys ¹⁵ -Ser ¹⁶ -PEG3 41

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Nil C4:C12, C7:C15 PEG3-Asn ⁱ -Asp ² -Glu ³ -Cys ⁴ -Glu ³ -Leu ⁶ -Cys ^/ -Val ⁸ -Asn ⁹ -Val ^iU -Ala ⁱⁱ -Cys ⁱ² -Thr ⁱ³ Gly ¹⁴ -Cys ¹⁵ -dSer ¹⁶ -PEG3 42 N12 C4:C12, C7:C15 PEG3-Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys ^/ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dSer ¹⁶ 43 N13 C4:C12,C7:C15 Asn ⁱ -Asp ² -Glu ³ -Cys ⁴ -Glu ³ -Leu ⁶ -Cys ^/ -Val ⁸ -Asn ⁹ -Val ^iU -Ala ⁱⁱ -Cys ⁱ² -Thr ⁱ³ -Gly ¹⁴ Cys ¹⁵ -dSer ¹⁶ -PEG3 44 Formula I C4:C12, C7:C15 Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Xaa ³ -Xaa ⁶ -Cys ^/ -Xaa ⁸ -Xaa ⁹ -Xaa ¹⁰ -Xaa ¹¹ -Cys ¹² -Xaa ¹³ -Xaa ¹⁴ - Cys ¹⁵ -Xaa ¹⁶ 45 Formula II C4:C12, C7:C15 Xaa _n i-Cys ⁴ -Xaa ^b -Xaa ⁶ -Cys ^/ -Xaa ⁸ -Xaa ⁹ -Xaa ¹⁰ -Xaa ¹¹ -Cys ¹² -Xaa ¹³ -Xaa ¹⁴ -Cys ^lb Xaa _n 2 ¹⁶ 46 Formula III 4:12,7:15 Xaa _n i-Maa ⁴ -Glu ³ -Xaa ⁶ -Maa ^/ -Val ⁸ -Asn ⁹ -Val ^iU -Ala ⁱⁱ -Maa ⁱ² -Thr ⁱ³ -Gly ⁱ⁴ -Maa ^ib Xaa _n2 47 Formula IV 4:12,7:15 Xaa _n rMaa ⁴ -Xaa ³ -Xaa ⁶ -Maa '-Xaa ⁸ -Xaa ⁹ -Xaa ¹⁰ -Xaa ⁿ -Maa ¹² -Xaa ¹³ -Xaa ¹⁴ -Maa ^lb - Xaa _n 2 48 Formula V C4:C12,C7:C15 Asn ¹ -Asp ² -Asp ³ -Cys ⁴ -Xaa ³ -Xaa ⁶ -Cys ^/ -Xaa ⁸ -Asn ⁹ -Xaa ^{lu -Xaa 11 -Cys 12 -Xaa 13 -Xaa 14 - Cys 15 -Xaa 16} 49 Formula VI C4:C12, C7:C15 dAsn ¹ -Glu ² -Glu ³ -Cys ⁴ -Xaa ³ -Xaa ⁶ -Cys ^/ -X3 ⁸ -Asn ⁹ -Xaa ^lu -Xaa ¹¹ -Cys ¹² -Xaa ¹³ -Xaa ¹⁴ - Cys ¹⁵ -d-Xaa ¹⁶ 50 Formula VII C4:C12, C7:C15 dAsn ¹ -dGlu ² -Asp ³ -Cys ⁴ -Xaa ³ -Xaa ⁶ -Cys ^/ -Xaa ⁸ -Asn 9 -Xaa ¹⁰ -Xaa ¹¹ -Cys ¹² -Xaa ¹³ - Xaa ¹⁴ -Cys ^{15 -d} -Xaa ¹⁶ 51 Formula VII C4:C12, C7:C15 dAsn ¹ -dAsp ² -Glu ³ -Cys ⁴ -Xaa ³ -Xaa ^{6 -Cys /} -Xaa ⁸ -Asn ⁹ -Xaa ^lu -Xaa ¹¹ -Cys ^{12 -Xaa 13} - Xaa ¹⁴ -Cys ¹⁵ -d-Xaa ¹⁶ 52

Formula VIII C4:C12,C7:C15 dAsn ⁱ -dAsp ² -dGlu ³ -Cys ⁴ -Xaa ³ -Xaa ⁶ -Cys ^/ -Xaa ⁸ -Tyr ⁹ -Xaa ^iU -Xaa ⁱⁱ -Cys ⁱ² -Xaa ⁱ³ - Xaa ¹⁴ -Cys ¹⁵ -d-Xaa ¹⁶ 53 Formula IX C4:C12,C7:C15 dAsn ⁱ -dGlu ² -dGlu ³ -Cys ⁴ -Xaa ³ -Xaa ⁶ -Cys ^/ -Xaa ⁸ -Tyr ⁹ -Xaa ^iU -Xaa ⁱⁱ -Cys ⁱ² -Xaa ⁱ³ Xaa ¹⁴ -Cys ¹⁵ -d-Xaa ¹⁶ 54 Formula XXI C4:C12,C7:C15 Xaa _n i-Cys ⁴ -Xaa ³ -Xaa ⁶ -Xaa ^/ -Xaa ⁸ -Xaa ⁹ -Xaa ¹⁰ -Xaa ¹¹ -Cys ¹² -Xaa ¹³ -Xaa ¹⁴ -Xaa ¹³ - Xaa _n2 ¹⁶ 250

Table II. Linaclotide and derivatives

Name Position of disulfide bonds Structure SEQ ID NO: SP-339 (linaclotide) C1:C6, C2:C10, C5:13 Cys ¹ -Cys ² -Glu3-Tyr ⁴ -Cys ³ -Cys ⁶ -Asn ^/ -Pro ⁸ -Ala ⁹ -Cys ¹⁰ -Thr ¹¹ -Gly ¹² - Cys ¹³ -Tyr ¹⁴ 55 SP-340 C1:C6, C2:C10, C5:13 Cys ⁱ -Cys ² -Glu ³ -Tyr ⁴ -Cys ³ -Cys ⁶ -Asn ^/ -Pro ⁸ -Ala ⁹ -Cys ^iU -Thr ⁱⁱ -Gly ⁱ² Cys ¹³ 56 SP-349 C1:C6, C2:C10, C5: 13 PEG3-Cys ¹ -Cys ² -Glu ³ -Tyr ⁴ -Cys ^b -Cys ⁶ -Asn ^/ -Pro ⁸ -Ala ⁹ -Cys ¹⁰ -Thr ¹¹ Gly ¹² -Cys ¹³ -Tyr ¹⁴ -PEG3 57 SP-353 C3:C8, C4:C12, C7 : 15 Asn ⁱ -Phe ² -Cys ³ -Cys ⁴ -Glu ³ -Ser ⁶ -Cys ^/ -Cys ⁸ -Asn ⁹ -Pro ^iU -Ala ⁱⁱ -Cys ⁱ² - Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Tyr ¹⁶ 58 SP-354 C3:C8, C4:C12, C7 : 15 AsrE-Phe^Cys^Cys^Gliy-Phe^Cys'-Cys^AsrE-Pro^-Ala^-Cys ¹² Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Tyr ¹⁶ 59

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SP-355 31:36, 32:310, 35:13 Cys ¹³ -dTyr ¹⁴ 60 SP-357 31:36, 32:310, 35:13 PEG3-3ys ⁱ -3ys ² -Glu ^J -Tyr ⁴ -3ys ^b -3ys ⁶ -Asn ^/ -Prc/-Ala ^y -3ys ^iU -Thr ⁱⁱ Gly ¹² -3ys ¹³ -Tyr ¹⁴ 61 SP-374 33:38, 34:312, 37:15 Asn ⁱ -Phe ² -Cys ³ -Cys ⁴ -Glu ^b -Thr ⁶ -Cys ^/ -Gys ³ -Asn ^y -Pro ^iU -Ala ⁱⁱ -Gys ⁱ² Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Tyr ¹⁶ 62 SP-375 33:38, 34:312, 37:15 Asn ⁱ -Phe ² -Cys ³ -Cys ⁴ -Glu ^b -Ser ⁶ -Cys ^/ -Gys ³ -Asn ^y -Pro ^iU -Ala ⁱⁱ -Gys ⁱ² Thr ¹³ -Gly ¹⁴ -Gys ¹⁵ -dTyr ¹⁶ 63 SP-376 33:38, 34:312, 37:15 dAsn ⁱ -Phe ² -Cys ³ -Cys ⁴ -Glu ^b -Ser ⁶ -Cys ^/ -Cys ³ -Asn ^y -Pro ^iU -Ala ⁱⁱ -Cys ⁱ² Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Tyr ¹⁶ 64 SP-377 33:38, 34:312, 37:15 dAsn ⁱ -Phe ² -Cys ³ -Cys ⁴ -Glu ^b -Ser ⁶ -Cys '-Cys^AsiZ-Pro^-Ala^-Cys ¹² Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dTyr ¹⁶ 65 SP-378 33:38, 34:312, 37:15 Asn ⁱ -Phe ² -Cys ³ -Cys ⁴ -Glu ^b -Thr ⁶ -Cys ^/ -Cys ³ -Asn ^y -Pro ^iU -Ala ⁱⁱ -Cys ⁱ² Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dTyr ¹⁶ 66 SP-379 33:38, 34:312, 37:15 dAsn ⁱ -Phe ² -Cys ³ -Cys ⁴ -Glu ^b -Thr ⁶ -Cys '-Cys^AsiZ-Pro^-Ala^-Cys ¹² Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Tyr ¹⁶ 67 SP-380 33:38, 34:312, 37:15 dAsn ⁱ -Phe ² -Cys ³ -Cys ⁴ -Glu ^b -Thr ⁶ -Cys ^/ -Cys ³ -Asn ^y -Pro ^iU -Ala ⁱⁱ -Cys ⁱ² Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dTyr ¹⁶ 68 SP-381 33:38, 34:312, 37:15 Asn ¹ -Phe ² -Gys ³ -Gys ⁴ -Glu ^b -Phe ⁶ -Gys ^/ -Gys ⁸ -Asn ⁹ -Pro ¹⁰ -Ala ¹¹ -Gys ¹² - Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dTyr ¹⁶ 69 SP-382 33:38, 34:312, 37:15 dAsn ⁱ -Phe ² -Cys ³ -Cys ⁴ -Glu ^b -Phe ⁶ -Cys '-Cys^AsiZ-Pro^-Ala^-Cys ¹² Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Tyr ¹⁶ 70 SP-383 33:38, 34:312, 37:15 dAsn ⁱ -Phe ² -Cys ³ -Cys ⁴ -Glu ^b -Phe ⁶ -Cys ^/ -Cys ⁸ -Asn ^y -Pro ^iU -Ala ⁱⁱ -Cys ⁱ² Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dTyr ¹⁶ 71 SP-384 31:36, 32:310, 35:13 Cys ⁱ -Cys ² -Glu ³ -Tyr ⁴ -Cys ^b -Cys ⁶ -Asn ^/ -Pro ⁸ -Ala ^y -Cys ^iU -Thr ⁱⁱ -Gly ⁱ² Cys ¹³ -Tyr ¹⁴ -PEG3 72 N14 31:36, 32:310, 35:13 PEG3-Cys ⁱ -Cys ² -Glu ³ -Tyr ⁴ -Cys ^b -Cys ⁶ -Asn ^/ -Pro ⁸ -Ala ^y -Cys ^iU -Thr ⁱⁱ Gly ¹² -Cys ¹³ -PEG3 73 N15 31:36, 32:310, 35:13 PEG3-Cys ¹ -Cys ² -Glu ³ -Tyr ⁴ -Cys ^b -Cys ⁶ -Asn ^/ -Pro ⁸ -Ala ⁹ -Cys ¹⁰ -Thr ¹¹ -Gly ¹² -Gys ¹³ 74 N16 31:36, 32:310, 35:13 Cys ⁱ -Cys ² -Glu ³ -Tyr ⁴ -Cys ^b -Cys ⁶ -Asn ^/ -Pro ⁸ -Ala ⁹ -Cys ^iU -Thr ⁱⁱ -Gly ⁱ² Cys ¹³ -PEG3 75 N17 33:38, 34:312, 37:15 PEG3-Asn ⁱ -Phe ² -Cys ³ -Cys ⁴ -Glu ^b -Ser ⁶ -Cys ^/ -Cys ⁸ -Asn ⁹ -Pro ^iU -Ala ⁱⁱ Cys ¹² -Thr 13 ^{-Gly 14} -Cys ^{15 -Tyr 16} -PEG3 76 N18 33:38, 34:312, 37:15 PEG3-Asn ¹ -Phe ² -Cys ³ -Cys ⁴ -Glu ^b -Ser ⁶ -Cys ^/ -Cys ⁸ -Asn ⁹ -Pro ¹⁰ -Ala ¹¹ - Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Tyr ¹⁶ 77 N19 33:38, 34:312, 37:15 Asn ⁱ -Phe ² -Cys ³ -Cys ⁴ -Glu ^b -Ser ⁶ -Cys ^/ -Cys ⁸ -Asn ⁹ -Pro ^iU -Ala ⁱⁱ -Cys ⁱ² - Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Tyr ¹⁶ -PEG3 78 N2 0 33:38, 34:312, C7:15 PEG3-Asn ¹ -Phe ² -Cys ³ -Cys ⁴ -Glu ^b -Phe ⁶ -Cys ^/ -Cys ⁸ -Asn ⁹ -Pro ¹⁰ -Ala ¹¹ Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Tyr ¹⁶ -PEG3 79 N21 C3:C8, C4:C12, C7:15 PEG3-Asn ⁱ -Phe ² -Gys ³ -Gys ⁴ -Glu ^b -Phe ⁶ -Gys ^/ -Gys ⁸ -Asn ⁹ -Pro ^iU -Ala ⁱⁱ Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Tyr ¹⁶ 80 N22 C3:C8, C4:C12, C7 : 15 Asn ⁱ -Phe ² -Cys ³ -Cys ⁴ -Glu ^b -Phe ⁶ -Cys ^/ -Cys ⁸ -Asn ⁹ -Pro ^iU -Ala ⁱⁱ -Cys ⁱ² Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Tyr ¹⁶ -PEG3 81

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N2 3 NW:S8, 04:012, 07:15 PEG3-Asn ¹ -Phe ² -Cys ³ -Cys ⁴ -Gli?-Tyr ⁶ -Cys '-Cys^Asn^Pro^-Ala ¹¹ - Cys ¹² -Thr 13 -Gly ¹⁴ -Cys ^{15 -Tyr 16} -PEG3 82 N2 4 03:08, 04:012, 07:15 PEG3-Asn ¹ -Phe ² -Cys ³ -Cys ⁴ -Glu ^b -Tyr ⁶ -Cys ^/ -Oys ⁸ -Asny-Pro ¹⁰ -Ala ¹¹ Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Tyr ¹⁶ 83 N2 5 03:08, 04:012, 07:15 Asn ¹ -Phe ² -Cys ³ -Cys ⁴ -Glu ^b -Tyr ⁶ -Cys ^/ -Cys ⁸ -Asn ⁹ -Pro ¹⁰ -Ala ¹¹ -Cys ¹² Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Tyr ¹⁶ -PEG3 84 N2 6 S1:S6, 02:010, S5:13 Cys ⁱ -Cys ² -Glu3-Ser ⁴ -Cys ^b -Cys ⁶ -Asn ^/ -Pro ⁸ -Ala ⁹ -Cys ^iU -Thr ⁱⁱ -Gly ⁱ² Cys ¹³ -Tyr ¹⁴ 85 N2 7 C1:C6, 02:010, 05:13 Cys ⁱ -Cys ² -Glu3-Phe ⁴ -Cys ^b -Cys ⁶ -Asn ^/ -Pro ⁸ -Ala ⁹ -Cys ^iU -Thr ⁱⁱ -Gly ⁱ² Cys ¹³ -Tyr ¹⁴ 86 N2 8 C1:C6, 02:010, 05:13 Cys ⁱ -Cys ² -Glu3-Ser ⁴ -Cys ^b -Cys ⁶ -Asn ^/ -Pro ⁸ -Ala ⁹ -Cys ^iU -Thr ⁱⁱ -Gly ⁱ² Cys ¹³ - 87 N2 9 C1:C6, 02:010, 05:13 Cys ⁱ -Cys ² -Glu3-Phe ⁴ -Cys ^b -Cys ⁶ -Asn ^/ -Pro ⁸ -Ala ⁹ -Cys ^iU -Thr ⁱⁱ -Gly ⁱ² Cys ¹³ 88 N30 1:6, 2:10, 5:13 Pen ¹ -Pen ² -Glu3-Tyr ⁴ -Pen ^b -Pen ⁶ -Asn ^/ -Pro ⁸ -Ala ⁹ -Pen ¹⁰ -Thr ¹¹ -Gly ¹² -Pen ¹³ -Tyr ¹⁴ 89 N31 1:6, 2:10, 5:13 Pen ¹ -Pen ² -Glu3-Tyr ⁴ -Per? -Pen ⁶ -Asn ^/ -Pro ⁸ -Ala ⁹ -Pen ¹⁰ -Thr ¹¹ -Gly ¹² - Pen ¹³ 90 Formula X 09:014, 010:018, 013:21 Xaa ¹ - Xaa ² ~ Xaa 3 - Xaa ^{4 - Xaa b} - Xaa ⁶ - Azp ^/ - Tyr ⁸ - SuzU-Suz ¹⁰ - Xaa ¹¹ - Tyr ¹² Cys ^{13 -} Cys ^{14 -} Xaa ¹⁵ - Xaa ¹⁶ - Xaa ¹⁷ - Cys ¹⁸ -Xaa ¹⁹ -Xaa ²⁰ -Cys ²¹ -Xaa ²² 91 Formula XI 09:014, 010:018, 013:21 Xaa ¹ -Xaa ² ~ Xaa ³ -Xaa ⁴ -Xaa ^b -Xaa ⁶ -Azp ^/ -RPe ⁸ -SuzU-Suz ¹⁰ -Xaa ¹¹ -Pe ¹² Cys ¹³ -Cys ¹⁴ -Xaa ¹⁵ -Xaa ¹⁶ -Xaa ¹⁷ -Cys ¹⁸ -Xaa ¹⁹ -Xaa ²⁰ -Cys ²¹ -Xaa ²² 92

Formula XII 03:08, 04:012, 07:15 Asn ⁱ -Phe ² -Cys ³ -Cys ⁴ -Xaa ³ -Phe ⁶ -Cys ^/ -Cys ⁸ -Xaa ⁹ -Xaa ^iU -Xaa ⁱⁱ Xys ⁱ² Xaa ³ -Xaa ¹⁴ ~Suz ¹⁵ -Xaa ¹⁶ 93 Formula XIII 3:8, 4:12, S:15 Asn ¹ -Phe ² -Pen ³ -Cys ⁴ -Xaa ^b -Phe ⁶ -Cys ^/_ Rep ⁸ -XaaU-Xaa ¹⁰ -Xaa ^11- Suz12-Xaa ¹³ -Xaa ¹⁴ ~Cys ¹⁵ -Xaa ¹⁶ 94 Formula XIV 3:8, 4:12, 7:15 AsgC-Pe^Maa^Maa^Xaa^-Xaa^Maa^Maa^XaaY-Xaa^-Xaa^Maa ¹² Xaa ¹³ -Xaa ¹⁴ Maa ¹⁵ -Xaa ¹⁶ 95 Formula XV 1:6, 2:10, 5:13 Maa ⁱ -Maa ² -Glu3-Xaa ⁴ -Maa ^b -Maa ⁶ -Asn ^/ -Pro ⁸ -Ala ⁹ -Maa ^iU -Thr ⁱⁱ -Gly ⁱ² Maa ¹³ -Tyr ¹⁴ 96 Formula XVI 1:6, 2:10, 5:13 Maa ¹ -Maa ² -61uZ-Xaa ⁴ -Maa ^b -Maa ⁶ -Azp ^/ -Pro ⁸ -A1aU-Maa ¹⁰ -Tg ¹¹ -61u ¹² - Maa ¹³ - 97 Formula XVII 1:6, 2:10, 5:13 Xaa _n3 -Maa ¹ -Maa ² -Xaa ³ -Xaa ⁴ -Maa ^b -Maa ⁶ -Xaa ^/ -Xaa ⁸ -Xaa ⁹ -Maa ¹⁰ -Xaa ¹¹ - Xaa ¹² -Maa ¹³ -Xaa _n2 98

Table III. GCRA peptides

Name Position of disulfide bonds Structure SEQ ID NO: SP-363 C4:C12, C7:C15 dAsn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys ^/ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dLeu-AMHfl ¹⁶ 99 SP-364 04:C12, C7:C15 dAsn ⁱ -Asp ² -Glu ³ -Cys ⁴ -Gli? -Leu ⁶ -Cys ^/ -Val ⁸ -Asn ⁹ -Val ^iU -Ala ⁱⁱ -Cys ⁱ² Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dSer ¹⁶ 100 SP-365 C4:C12, C7:C15 dAsn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys ^/ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dSer-AMHfl ¹⁶ 101 SP-366 C4:C12, C7:C15 dAsn ⁱ -Asp ² -Glu ³ -Cys ⁴ -Gli? -Leu ⁶ -Cys ^/ -Val ⁸ -Asn ⁹ -Val ^iU -Ala ⁱⁱ -Cys ⁱ² Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dTyr ¹⁶ 102 SP-367 C4:C12, C7:C15 dAsn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys ^/ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² - Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dTyr-AMHfl ¹⁶ 103 SP-373 C4:C12, C7:C15 Pyglu ⁱ -Asp ² -Glu ³ -Cys ⁴ -Glu ³ -Leu ⁶ -Cys ^/ -Val ⁸ -Asn ⁹ -Val ^iU -Ala ⁱⁱ -Cys ⁱ² - Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dLeu-AMHfl ¹⁶ 104 / C4:C12, C7:C15 Pyglu ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys ^/ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Leu ¹⁶ 251 SP-304 di PEG C4:C12, C7:C15 PEGS-Asr^-Asp^GW-Cys^GW-Leu^Cys'-VaV-Asny-Val^-Ala^-Cys ¹² Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Leu ¹⁶ ~PEG3 105

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SP-304 N-PEG S4:012, 07:015 PEG3-Asn ¹ -Asp ² -Glu ³ -Oys ⁴ -Glu ^b -Leu ⁶ -Oys ^/ -Val ⁸ -Asn ⁹ -Var°-Ala ¹¹ -Cys ¹² Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Leu ¹⁶ 106 SP-304 C-PEG С4:С12, 07:015 Asn ⁱ -Asp ² -Glu ³ -Cys ⁴ -Glu ³ -Leu ⁶ -Cys ^/ -Val ⁸ -Asn ⁹ -Val ^iU -Ala ⁱⁱ -Cys ⁱ² -Thr ⁱ³ -Gly ¹⁴ -Oys ¹⁵ -Leu ¹⁶ ~PEG3 107

Table IV. SP-304 analogs, uroguaniline and uroguaniline analogs

Name Position of disulfide bonds Structure SEQ ID NO Formula XVIII 04:S12, 07:015 Xaa ¹ - Xaa ² - Xaa ³ - Maa ⁴ - ^{Xaa b} - Xaa ⁶ - Maa ^/ - Xaa ⁸ - Xaa ⁹ - Xaa ¹⁰ - Xaa ¹¹ - Maa ¹² - Xaa ¹³ - Xaa ¹⁴ - Maa ¹⁵ - Xaa ¹⁶ 108 Urogua- nilin 04:S12, 07:015 Asn ⁱ -Asp ² -Asp ³ -Cys ⁴ -Glu ³ -Leu ⁶ -Cys '-Var-AsrT-Val^-Ala^-Cys^-Thr^-Gly ¹⁴ Cys ¹⁵ -Leu ¹⁶ 109 N32 04:S12, 07:015 Glu ⁱ -Asp ² -Asp ³ -Cys ⁴ -Glu ³ -Leu ⁶ -Cys ^/ -Var-Asn ⁹ -Val ^iU -Ala ⁱⁱ -Cys ⁱ² -Thr ⁱ³ -Gly ⁱ⁴ Cys ¹⁵ -Leu ¹⁶ 110 N33 04:S12, 07:015 Glu ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys ^/ -Val ⁸ -Asn ⁹ -Var°-Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ Cys ¹⁵ -Leu ¹⁶ 111 N34 С4:С12, 07:015 Glu ¹ -Glu ² -Asp ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys ^/ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ - Cys ¹⁵ -Leu ¹⁶ 112 N35 С4:С12, 07:015 Glu ¹ -Glu ² -Glu ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys ^/ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ - Cys ¹⁵ -Leu ¹⁶ 113 N3 6 04:S12, 07:015 Asp ⁱ -Asp ² -Asp ³ -Cys ⁴ -Glu ³ -Leu ⁶ -Cys ^/ -Val ⁸ -Asn ⁹ -Val ^iU -Ala ⁱⁱ -Cys ⁱ² -Thr ⁱ³ -Gly ⁱ⁴ Cys ¹⁵ -Leu ¹⁶ 114 N37 04:S12, 07:015 Asp ⁱ -Asp ² -Glu ³ -Cys ⁴ -Glu ³ -Leu ⁶ -Cys ^/ -Var-Asn ⁹ -Val ^iU -Ala ⁱⁱ -Cys ⁱ² -Thr ⁱ³ -Gly ⁱ⁴ Cys ¹⁵ -Leu ¹⁶ 115 N38 04:S12, 07:015 Asp ¹ -Glu ² -Asp ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys ^/ -Val ⁸ -Asn ⁹ -Var°-Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ Cys ¹⁵ -Leu ¹⁶ 116 N39 04:S12, 07:015 Asp ¹ -Glu ² -Glu ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys ^/ -Val ⁸ -Asn ⁹ -Var°-Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ Cys ¹⁵ -Leu ¹⁶ 117 N4 0 04:012, 07:015 Gln ⁱ -Asp ² -Asp ³ -Cys ⁴ -Glu ³ -Leu ⁶ -Cys ^/ -Var-Asn ⁹ -Val ^iU -Ala ⁱⁱ -Cys ⁱ² -Thr ⁱ³ -Gly ⁱ⁴ Cys ¹⁵ -Leu ¹⁶ 118 N41 04:012, 07:015 Gln ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ³ -Leu ⁶ -Cys ^/ -Val ⁸ -Asn ⁹ -Val ^lu -Ala ¹¹ -Cys ^{12 -Thr 13} -Gly ¹⁴ - Cys ¹⁵ -Leu ¹⁶ 119 N42 04:012, 07:015 Gln ¹ -Glu ² -Asp ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys ^/ -Val ⁸ -Asn ⁹ -Var°-Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ Cys ¹⁵ -Leu ¹⁶ 120 N43 04:012, 07:015 Gln ¹ -Glu ² -Glu ³ -Cys ⁴ -Glu ³ -Leu ⁶ -Cys ^/ -Val ⁸ -Asn ⁹ -Val ^lu -Ala ¹¹ -Cys ^{12 -Thr 13} -Gly ¹⁴ - Cys ¹⁵ -Leu ¹⁶ 121 N44 04:012, 07:015 Lys ⁱ -Asp ² -Asp ³ -Cys ⁴ -Glu ³ -Leu ⁶ -Cys ^/ -Val ⁸ -Asn ⁹ -Val ^iU -Ala ⁱⁱ -Cys ⁱ² -Thr ⁱ³ -Gly ⁱ⁴ - Cys ¹⁵ -Leu ¹⁶ 122 N45 04:012, 07:015 Lys ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys ^/ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ - Cys ¹⁵ -Leu ¹⁶ 123 N4 6 04:012, 07:015 Lys ¹ -Glu ² -Asp ³ -Cys ⁴ -Glu ³ -Leu ⁶ -Cys ^/ -Val ⁸ -Asn ⁹ -Val ^lu -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ - Cys ¹⁵ -Leu ¹⁶ 124

- 28 042294

N47 04:012, 07:015 1.73^611^-61^-073^611^-1.31^-073^31^33^31^^13^-073^-1113^-617^Cys ¹⁵ -Leu ¹⁶ 125 N4 8 04:012, 07:015 61u ¹ -Asp ² -Asp ³ -C7S ⁴ -61u ^b -Leu ⁶ -C7S ^/ -V31 ⁸ -Asn ⁹ -V31 ¹⁰ -A13 ¹¹ -07S ¹² -Thr ¹³ -617 ¹⁴ C73 ¹⁵ -Ser ¹⁶ 126 N4 9 04:012, 07:015 6lu ⁱ -Asp ² -6lu ³ -C7S ⁴ -6lu ³ -Leu ^b -C7S ^z -V3l ⁸ -As3 ^y -V3l ^iU -Al3 ⁱⁱ -C7S ⁱ² -1hr ⁱ³ -6l7 ⁱ⁴ C73 ¹⁵ -Ser ¹⁶ 127 N50 04:012, 07:015 6lu ⁱ -6lu ² -Asp ³ -C7S ⁴ -6lu ³ -Leu ^b -C7S ^z -V3l ⁸ -As3 ^y -V3l ^iU -Al3 ⁱⁱ -C7S ⁱ² -1hr ⁱ³ -6l7 ⁱ⁴ C7s ¹⁵ -Ser ¹⁶ 128 N51 04:012, 07:015 61u ¹ -61u ² -61u ³ -C7S ⁴ -61u ^b -Leu ⁶ -C7S ^/ -V31 ⁸ -Asn ^y -V31 ¹⁰ -A13 ¹¹ -07S ¹² -Thr ¹³ -617 ¹⁴ C73 ¹⁵ -Ser ¹⁶ 129 N52 04:012, 07:015 Asp ⁱ -Asp ² -Asp ³ -C7S ⁴ -6lu ³ -Leu ⁶ -C7S ^z -V3l ⁸ -As3 ^y -V3l ^iU -Al3 ⁱⁱ -07S ⁱ² -1hr ⁱ³ -6l7 ⁱ⁴ C73 ¹⁵ -Ser ¹⁶ 130 N53 04:012, 07:015 Asp ¹ -Asp ² -61u ³ -C7S ⁴ -61u ^b -Leu ⁶ -07S ^/ -V31 ⁸ -Asn ⁹ -V31 ¹⁰ -A13 ¹¹ -C7S ¹² -Thr ¹³ -617 ¹⁴ C73 ¹⁵ -Ser ¹⁶ 131 N54 04:012, 07:015 Asp ¹ -61u ² -Asp ³ -C7S ⁴ -61u ^b -Leu ⁶ -07S ^/ -V31 ⁸ -Asn ⁹ -V31 ¹⁰ -A13 ¹¹ -C7S ¹² -Thr ¹³ -617 ¹⁴ C73 ¹⁵ -Ser ¹⁶ 132 N55 04:012, 07:015 Asp ⁱ -6lu ² -6lu ³ -C7S ⁴ -6lu ³ -Leu ^b -C7S ^z -V3l ⁸ -As3 ^y -V3l ^iU -Al3 ⁱⁱ -C7S ⁱ² -1hr ⁱ³ -6l7 ⁱ⁴ C73 ¹⁵ -Ser ¹⁶ 133 N56 04:C12, 07:015 613 ¹ -Asp ² -Asp ³ -C7S ⁴ -61u ^b -Leu ⁶ -07S ^1^3^^33^1^31^^13^-073^-1113^-617^- C73 ¹⁵ -Ser ¹⁶ 134 N57 04:012, 07:015 61n ⁱ -Asp ² -61u ³ -C7S ⁴ -61u ³ -Leu ^b -C7S ^/ -V31 ⁸ -Asn ^{9 -V31 iU} -A13 ⁱⁱ -C7S ⁱ² -Thr ⁱ³ -617 ⁱ⁴ C73 ¹⁵ -Ser ¹⁶ 135

N58 04:012, 07:015 6l3 ⁱ -6lu ² -Asp ³ -C7S ⁴ -6lu ³ -Leu ⁶ -C7S ^z -V3l ⁸ -As3 ^y -V3l ^iU -Al3 ⁱⁱ -C7S ⁱ² -1h3 i3 -6l7 ^{i4 C73 15} -Ser ¹⁶ 136 N59 04:012, 07:015 611^-611^-61^-073^611^-1.31^-073^31^33^31^^13^-073^-1111:^-617^Cys ¹⁵ -Ser ¹⁶ 137 N60 04:012, 07:015 Lys ¹ -Asp ² -Asp ³ -C7s ⁴ -611? -Leu ⁶ -C7s ^31^33^31^^13^-073^-1111:^-617^- C73 ¹⁵ -Ser ¹⁶ 138 N61 04:012, 07:015 Lys ⁱ -Asp ² -61u ³ -C7S ⁴ -61u ³ -Leu ^b -C7S ^Z -Vsl ⁸ -As3 ⁹ -Vsl ^iU -Al3^-673 ⁱ² -1hr ⁱ³ -6l7 ⁱ⁴ - Cys ¹⁵ -Ser ¹⁶ 139 N62 04:012, 07:015 L7s ¹ -61u ² -Asp ³ -C73 ⁴ -61u ^{; >} -Leu ⁶ -C7S ^Z -V31 ⁸ -Azz ^y -Uz1 ^1u -A13^-673 ^l2 -1hr ^l3 -6l7 ¹⁴ - C73 ¹⁵ -Ser ¹⁶ 140 N63 04:012, 07:015 L7s ¹ -61u ² -61u ³ -C7S ⁴ -61u ^b -Leu ^b -07S ^z -V3l ^{8 -As3 9} -V3l ^lu -Al3 ^ll -C7S ^l2 -1hr ^l3 -6l7 ¹⁴ - C73 ¹⁵ -Ser ¹⁶ 141 N65 04:012, 07:015 6lu ⁱ -Asp ² -Asp ³ -C7S ⁴ -6lu ³ -Leu ⁶ -C7S ^z -Ile ⁸ -As3 ⁹ -Met ^iU -Al3 ⁱⁱ -07S ⁱ² -1hr ⁱ³ -6l7 ⁱ⁴ C7S ¹⁵ -Leu ¹⁶ 142 N66 04:012, 07:015 6lu ^l -Asp ² -6lu ³ -C7S ⁴ -6lu ^b -Leu ⁶ -C7S ^Z -Ile ⁸ -As3 ⁹ -Met ^lo -Al3 ^ll -07S ^l2 -1hr ^l3 -6l7 ¹⁴ C7S ¹⁵ -Leu ¹⁶ 143 N67 04:012, 07:015 6lu ^l -6lu ² -Asp ³ -C7S ⁴ -6lu ^b -Leu ⁶ -C7S ^Z -Ile ⁸ -As3 ⁹ -Met ^lo -Al3 ^ll -07S ^l2 -1hr ^l3 -6l7 ¹⁴ Cys ¹⁵ -Leu ¹⁶ 144 N68 04:012, 07:015 61u ⁱ -61u ² -61u ³ -07S ⁴ -61u ³ -Leu ^b -C7S ^z -1le ⁸ -As3 ⁹ -Met ^iU -Al3 ⁱⁱ -C7S ⁱ² -1hr ⁱ³ -6l7 ⁱ⁴ - C7S ¹⁵ -Leu ¹⁶ 145 N69 04:012, 07:015 Α3ρ ² -Α3ρ ² -Α3ρ ³ -073 ⁴ -61υ ³ -Ι,θ3 ⁶ -073 ^ζ -Ι1θ ⁸ -Α33 ⁹ -Μθ1 ^ιυ -Α13 ²¹ -073 ¹² -11ΐ3 ¹³ -617 ¹⁴ C7S ¹⁵ -Leu ¹⁶ 146

- 29 042294

N70 04:012, 07:015 Asp^Asp^Glu^Cys^Glu^-Leu^Cys'-Ile^Asny-Met^-Ala^-Cys^-Thr^-Gly ¹⁴ Cys ¹⁵ -Leu ¹⁶ 147 N71 04:012, 07:015 Asp ⁱ -Glu ² -Asp ^J -Cys ⁴ -Glu ^b -Leu ⁶ -Cys '-Ile^Asny-Met^-Ala^-Cys^-Thr^-Gly ¹⁴ Cys ¹⁵ -Leu ¹⁶ 148 N72 04:012, 07:015 Asp ¹ -Glu ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ - Cys ¹⁵ -Leu ¹⁶ 149 N73 04:012, 07:015 Gln ¹ -Asp ² -Asp ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys '-iW-Asny-Met^-Ala^-Cys^-Thr^-Gly ¹⁴ Cys ¹⁵ -Leu ¹⁶ 150 N74 04:012, 07:015 Gln ⁱ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys '-Ile^Asny-Met^-Ala^-Cys^-Thr^-Gly ¹⁴ Cys ¹⁵ -Leu ¹⁶ 151 N75 04:012, 07:015 Gln ¹ -Glu ² -Asp ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys '-iW-Asny-Met^-Ala^-Cys^-Thr^-Gly ¹⁴ Cys ¹⁵ -Leu ¹⁶ 152 N7 6 04:012, 07:015 GW-GW-GW-Cys^Gluy-Leu^Cys'-Ile^Asny-Met^-Ala^-Cys^-Thr^-Gly ¹⁴ Cys ¹⁵ -Leu ¹⁶ 153 N77 04:012, 07:015 Lys ¹ -Asp ² -Asp ³ -Cys ⁴ -Glu ³ -Leu ^b -Cys ^/ -Ile ⁸ -Asn ^y -Met ^lu -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ - Cys ¹⁵ -Leu ¹⁶ 154 N7 8 04:012, 07:015 Lys ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys '-iW-Asny-Met^-Ala^-Cys^-Thr^-Gly ¹⁴ Cys ¹⁵ -Leu ¹⁶ 155 N79 04:012, 07:015 Lys ¹ -Glu ² -Asp ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys '-iW-Asny-Met^-Ala^-Cys^-Thr^-Gly ¹⁴ Cys ¹⁵ -Leu ¹⁶ 156 N80 04:012, 07:015 Lys ⁱ -Glu ² -Glu ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys '-Ile^Asny-Met^-Ala^-Cys^-Thr^-Gly ¹⁴ Cys ¹⁵ -Leu ¹⁶ 157 N81 04:012, 07:015 Glu ⁱ -Asp ² -Asp ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys'-Ile^Asny-Met^-Ala^-Cys^-Thr^-Gly ¹⁴ Cys ¹⁵ -Ser ¹⁶ 158 N82 04:012, 07:015 Glu ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys'-Ile^Asny-Met^-Ala^-Cys^-Thr^-Gly ¹⁴ Cys ¹⁵ -Ser ¹⁶ 159 N83 04:012, 07:015 Glu ⁱ -Glu ² -Asp ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys'-Ile^Asny-Met^-Ala^-Cys^-Thr^-Gly ¹⁴ Cys ¹⁵ -Ser ¹⁶ 160 N84 04:012, 07:015 Glu ¹ -Glu ² -Glu ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys ^/ -Ile ⁸ -Asny-Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ - Cys ¹⁵ -Ser ¹⁶ 161 N85 04:012, 07:015 Asp ⁱ -Asp ² -Asp ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys'-Ile^Asny-Met^-Ala^-Cys^-Thr^-Gly ¹⁴ Cys ¹⁵ -Ser ¹⁶ 162 N86 04:012, 07:015 Asp ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys'-Ile^Asny-Met^-Ala^-Cys^-Thr^-Gly ¹⁴ Cys ¹⁵ -Ser ¹⁶ 163 N87 04:012, 07:015 Asp ⁱ -Glu ² -Asp ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys'-Ile^Asny-Met^-Ala^-Cys^-Thr^-Gly ¹⁴ Cys ¹⁵ -Ser ¹⁶ 164 N88 04:012, 07:015 Asp ⁱ -Glu ² -Glu ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys'-Ile^Asny-Met^-Ala^-Cys^-Thr^-Gly ¹⁴ Cys ¹⁵ -Ser ¹⁶ 165 N89 04:012, 07:015 Gln ⁱ -Asp ² -Asp ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys ^/ -Ile ⁸ -Asn ^y -Met ^iU -Ala ⁱⁱ -Cys ⁱ² -Thr ¹³ -Gly ⁱ⁴ Cys ¹⁵ -Ser ¹⁶ 166 N90 04:012, 07:015 Gln ⁱ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys ^/ -Ile ⁸ -Asny-Met ^iU -Ala ⁱⁱ -Cys ⁱ² -Thr ¹³ -Gly ⁱ⁴ Cys ¹⁵ -Ser ¹⁶ 167 N91 04:012, 07:015 Gln ¹ -Glu ² -Asp ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys'-Ile^Asny-Met^-Ala^-Cys^-Thr^-Gly ¹⁴ Cys ¹⁵ -Ser ¹⁶ 168

N92 04:012, 07:015 Gln ⁱ -Glu ² -Glu ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys ^/ -Ile ⁸ -Asn ^y -Met ^iU -Ala ⁱⁱ -Cys ⁱ² -Thr ⁱ³ -Gly ⁱ⁴ Cys ¹⁵ -Ser ¹⁶ 169 N93 04:012, 07:015 Lys ⁱ -Asp ² -Asp ³ -Cys ⁴ -Gli? -Leu ⁶ -Cys ^/ -Ile ⁸ -Asn ^y -Met ^iU -Ala ⁱⁱ -Cys ⁱ² -Thr ⁱ³ -Gly ⁱ⁴ Cys ¹⁵ -Ser ¹⁶ 170 N94 04:012, 07:015 Lys ⁱ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys ^/ -Ile ⁸ -Asn ^y -Met ^iU -Ala ⁱⁱ -Cys ⁱ² -Thr ⁱ³ -Gly ⁱ⁴ Cys ¹⁵ -Ser ¹⁶ 171 N95 04:012, 07:015 Lys ⁱ -Glu ² -Asp ³ -Cys ⁴ -Gli? -Leu ⁶ -Cys ^/ -Ile ⁸ -Asn ^y -Met ^iU -Ala ⁱⁱ -Cys ⁱ² -Thr ⁱ³ -Gly ⁱ⁴ Cys ¹⁵ -Ser ¹⁶ 172 N96 04:012, 07:015 Lys ⁱ -Glu ² -Glu ³ -Cys ⁴ -Gli? -Leu ⁶ -Cys ^/ -Ile ⁸ -Asn ^y -Met ^iU -Ala ⁱⁱ -Cys ⁱ² -Thr ⁱ³ -Gly ⁱ⁴ Cys ¹⁵ -Ser ¹⁶ 173

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Table V. Guaniline and analogues

Name- ing Position of disulfide bonds Structure SEQ ID NO Formula XIX 4:12,7:15 Xaa ¹ -Xaa ² -Xaa ³ -Maa ⁴ -Xaa ^b -Xaa ⁶ -Maa ^/ -Xaa ⁸ -Xaa ⁹ -Xaa ¹⁰ -Xaa ¹¹ -Maa ¹² -Xaa ¹³ - Xaa ¹⁴ -Maa ¹⁵ 174 Guanylin 04:012, 07:015 Ser ⁱ -His ² -Thr ³ -0ys ⁴ -Glu ^b -Ile ⁶ -Cys ^/ -Ala ⁸ -Phe ⁹ -Ala ^iU -Ala ⁱⁱ -0ys ⁱ² -Ala ⁱ³ Gly ¹⁴ -Cys ¹⁵ 175 Guanylin human 04:012, 07:015 Pro ⁱ -Gly ² -Thr ³ -Cys ⁴ -Glu ^b -Ile ⁶ -Cys ^/ -Ala ⁸ -Tyr ⁹ -Ala ^iU -Ala ⁱⁱ -Cys ⁱ² -Thr ⁱ³ Gly ¹⁴ -Cys ¹⁵ 252 N97 04:012, 07:015 Ser ⁱ -His ² -Thr ³ -Cys ⁴ -Glu ³ -Ile ⁶ -Cys ^/ -Ala ⁸ -Asn ^y -Ala ^iU -Ala ⁱⁱ -Cys ⁱ² -Ala ⁱ³ Gly ¹⁴ -Cys ¹⁵ 176 N98 04:012, 07:015 Ser ⁱ -His ² -Thr ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys ^/ -Ala ⁸ -Asn ⁹ -Ala ^iU -Ala ⁱⁱ -Cys ⁱ² -Ala ⁱ³ Gly ¹⁴ -Cys ¹⁵ 177 N99 04:012, 07:015 Ser ⁱ -His ² -Thr ³ -0ys ⁴ -Glu ^b -Val ⁶ -Cys ^/ -Ala ⁸ -Asn ⁹ -Ala ^iU -Ala ⁱⁱ -0ys ⁱ² -Ala ⁱ³ Gly ¹⁴ -Cys ¹⁵ 178 N100 04:012, 07:015 Ser ⁱ -His ² -Thr ³ -Cys ⁴ -Glu ³ -Tyr ⁶ -Cys ^/ -Ala ⁸ -Asn ^y -Ala ^iU -Ala ⁱⁱ -Cys ⁱ² -Ala ⁱ³ Gly ¹⁴ -Cys ¹⁵ 179 N101 С4:С12, 07:015 Ser ¹ -His ² -Thr ³ -Cys ⁴ -Glu ^b -Ile ⁶ -Cys ^/ -Ala ⁸ -Asn ⁹ -Ala ¹⁰ -Ala ¹¹ -Oys ¹² -Ala ¹³ -Gly ¹⁴ -Cys ¹⁵ 180 N102 04:012, 07:015 Ser ¹ -His ² -Thr ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys ^/ -Ala ⁸ -Asn ⁹ -Ala ¹⁰ -Ala ¹¹ -Cys ¹² -Ala ¹³ -Gly ¹⁴ -Cys ¹⁵ 181 N103 04:012, 07:015 Ser ¹ -His ² -Thr ³ -Cys ⁴ -Glu ^b -Val ⁶ -Cys ^/ -Ala ⁸ -Asn ⁹ -Ala ¹⁰ -Ala ¹¹ -Cys ¹² -Ala ¹³ -Gly ¹⁴ -Cys ¹⁵ 182 N104 04:012, 07:015 Ser ⁱ -His ² -Thr ³ -Cys ⁴ -Glu ³ -Tyr ⁶ -Cys ^/ -Ala ⁸ -Asn ⁹ -Ala ^iU -Ala ⁱⁱ -Cys ⁱ² -Ala ⁱ³ Gly ¹⁴ -Cys ¹⁵ 183 N105 04:012, 07:015 Ser ⁱ -His ² -Thr ³ -Cys ⁴ -Glu ³ -Ile ⁶ -Cys ^/ -Ala ⁸ -Asn ⁹ -Ala ^iU -Ala ⁱⁱ -Cys ⁱ² -Ala ⁱ³ Gly ¹⁴ -Cys ¹⁵ 184 N106 С4:С12, 07:015 Ser ⁱ -His ² -Thr ³ -Cys ⁴ -Glu ³ -Leu ⁶ -Cys ^/ -Ala ⁸ -Asn ⁹ -Ala ^iU -Ala ⁱⁱ -Cys ⁱ² -Ala ⁱ³ Gly ¹⁴ -Cys ¹⁵ 185 N107 04:012, С7:С15 Ser ⁱ -His ² -Thr ³ -Cys ⁴ -Glu ^b -Val ⁶ -Cys ^/ -Ala ⁸ -Asn ⁹ -Ala ^iU -Ala ⁱⁱ -Cys ⁱ² -Ala ⁱ³ Gly ¹⁴ -Cys ¹⁵ 186

N108 04:C12, 07:015 Ser ⁱ -His ² -Thr ³ -Cys ⁴ -Glu ^b -Tyr ⁶ -Cys ^/ -Ala ⁸ -Asn ⁹ -Ala ^iU -Ala ⁱⁱ -Cys ⁱ² -Ala ⁱ³ -Gly ¹⁴ -Cys ¹⁵ 187 N109 04:C12, 07:015 Ser ⁱ -His ² -Thr ³ -Cys ⁴ -Glu ³ -Ile ⁶ -Cys ^/ -Ala ⁸ -Asn ⁹ -Ala ^iU -Ala ⁱⁱ -Cys ⁱ² -Ala ⁱ³ Gly ¹⁴ -Cys ¹⁵ 188 N110 04:C12, 07:015 Ser ⁱ -His ² -Thr ³ -0ys ⁴ -Glu ³ -Leu ⁶ -Cys ^/ -Ala ⁸ -Asn ⁹ -Ala ^iU -Ala ⁱⁱ -0ys ⁱ² -Ala ⁱ³ Gly ¹⁴ -Cys ¹⁵ 189 Nlll 04:012, 07:015 Ser ¹ -His ² -Thr ³ -Cys ⁴ -Glu ^b -Var-Cys ^/ -Ala ⁸ -Asn ⁹ -Ala ¹⁰ -Ala ¹¹ -Cys ¹² -Ala ¹³ - Gly ¹⁴ -Cys ¹⁵ 190 N112 04:012, 07:015 Ser ¹ -His ² -Thr ³ -Cys ⁴ -Glu ^b -Tyr ⁶ -Cys ^/ -Ala ⁸ -Asn ⁹ -Ala ¹⁰ -Ala ¹¹ -Cys ¹² -Ala ¹³ -Gly ¹⁴ -Cys ¹⁵ 191 N113 04:012, 07:015 Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -Ile ⁶ -Cys ^/ -Ala ⁸ -Asn ⁹ -Ala ¹⁰ -Ala ¹¹ -Cys ¹² -Ala ¹³ -Gly ¹⁴ -Cys ^lb 192 N114 04:012, 07:015 Azp ² -Azr ² -61i ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys ^/ -Ala ⁸ -Asn ⁹ -Ala ^iU -Ala ⁱⁱ -Cys ⁱ² -Ala ⁱ³ Gly ¹⁴ -Cys ^lb 193 N115 04:012, 07:015 Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -Val ⁶ -Cys ^/ -Ala ⁸ -Asn ⁹ -Ala ^lu -Ala ¹¹ -Cys ¹² -Ala ¹³ -Gly ¹⁴ -Cys ^lb 194 N116 04:012, 07:015 Asn ⁱ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -Tyr ⁶ -Cys ^/ -Ala ⁸ -Asn ⁹ -Ala ^iU -Ala ⁱⁱ -Cys ⁱ² -Ala ⁱ³ Gly ¹⁴ -Cys ^lb 195 N117 04:012, 07:015 Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -Ile ⁶ -Cys ^/ -Ala ⁸ -Asn ⁹ -Ala ¹⁰ -Ala ¹¹ -Oys ¹² -Ala ¹³ -Gly ¹⁴ -Cys ^lb 196 N118 04:012, 07:015 Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys ^/ -Ala ⁸ -Asn ⁹ -Ala ^lu -Ala ¹¹ -Cys ¹² -Ala ¹³ -Gly ¹⁴ -Cys ^lb 197

- 31 042294

N119 C4:C12, C7:C15 Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ³ -Val ⁶ -Cys '-Ala^AsrT-Ala^-Ala^-Cys^-Ala ¹³ Gly ¹⁴ -Cys ¹⁵ 198 N120 C4:C12, C7:C15 Asn ⁱ -Asp ³ -Glu ³ -Cys ⁴ -Glu ³ -Tyr ⁶ -Cys ^/ -Ala ⁸ -Asn ⁹ -Ala ^iU -Ala ⁱⁱ -Cys ⁱ² -Ala ⁱ³ Gly ¹⁴ -Cys ¹⁵ 199 N121 C4:C12, C7:C15 Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -Ile ⁶ -Cys ^/ -Ala ⁸ -Asn ⁹ -Ala ¹⁰ -Ala ¹¹ -Cys ¹² -Ala ¹³ -Gly ¹⁴ -Cys ¹⁵ 200 N122 C4:C12, C7:C15 Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ³ -Leu ⁶ -Cys ^/ -Ala ⁸ -Asn ⁹ -Ala ^lu -Ala ¹¹ -Cys ¹² -Ala ¹³ -Gly ¹⁴ -Cys ¹⁵ 201 N123 C4:C12, C7:C15 Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ³ -Val ⁶ -Cys ^/ -Ala ⁸ -Asn ⁹ -Ala ^lu -Ala ¹¹ -Cys ¹² -Ala ¹³ Gly ¹⁴ -Cys ¹⁵ 202 N124 C4:C12, C7:C15 Azp ² -Azr ² -61i ³ -Suz ⁴ -61i ³ -Tug ^b -Suz ^/ -Ala ⁸ -Asn ⁹ -Ala ^iU -Ala ⁱⁱ -Cys ⁱ² -Ala ⁱ³ - Gly ¹⁴ -Cys ¹⁵ 203 N125 C4:C12, C7:C15 Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -Ile ⁶ -Cys ^/ -Ala ⁸ -Asn ⁹ -Ala ¹⁰ -Ala ¹¹ -Cys ¹² -Ala ¹³ -Gly ¹⁴ -Cys ¹⁵ 204 N126 C4:C12, C7:C15 Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -Leu ⁶ -Cys ^/ -Ala ⁸ -Asn ⁹ -Ala ¹⁰ -Ala ¹¹ -Cys ¹² -Ala ¹³ -Gly ¹⁴ -Cys ¹⁵ 205 N127 C4:C12, C7:C15 Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ³ -Val ⁶ -Cys ^/ -Ala ⁸ -Asn ⁹ -Ala ^lu -Ala ¹¹ -Cys ¹² -Ala ¹³ -Gly ¹⁴ -Cys ¹⁵ 206 N128 C4:C12, C7:C15 Azp ² -Azr ² -61i ³ -Suz ⁴ -61i ³ -Tug ^b -Suz ^/ -Ala ⁸ -Asn ⁹ -Ala ^iU -Ala ⁱⁱ -Cys ⁱ² -Ala ⁱ³ - Gly ¹⁴ -Cys ¹⁵ 207

Table VI. Lymphoguaniline and analogues

Name Position of disulfide bonds Structure SEQ ID NO Formula XX 4:12,7:15 Xaa ¹ -Xaa ² -Xaa ³ -Maa ⁴ -Xaa ³ -Xaa ⁶ -Maa ^/ -Xaa ⁸ -Xaa ⁹ -Xaa ¹ⁱ -Xaa 11 -Maa ¹² -Xaa ¹³ - Xaa ^{14 -Xaa} _n i ¹⁵ 208 Lymphoguaniline C4:C12 Gln ¹ -Glu ² -Glu- ³ Cys ⁴ -Glu ^b -Leu ⁶ -Cys ^/ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ^{12 -Thr 13} -Gly ¹⁴ -Tyr ¹⁵ 209 N129 C4:C12 Gln ¹ -Glu ² -Glu ³ -Cys ⁴ -Glu ^b -Thr ⁶ -Cys ^/ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Tyr ¹⁵ 210 N130 C4:C12 Gln ⁱ -Asp ² -Glu ³ -Cys ⁴ -Glu ³ -Thr ⁶ -Cys ^/ -Ile ⁸ -Asn ⁹ -Met ^iU -Ala ⁱⁱ -Cys ⁱ² -Thr ⁱ³ Gly ¹⁴ -Tyr ¹⁵ 211 N131 C4:C12 C1p ² -Azr ² -Azr ³ -Cuz ⁴ -C1u ³ -TPg ⁶ -Cuz ^z -11e ⁸ -Azp ^y -Me5 ^1u -A1a ²¹ -Cuz ¹² -TPg ¹³ - Gly ¹⁴ -Tyr ¹⁵ 212 N132 C4:C12 Gln ¹ -Glu ² -Asp ³ -Cys ⁴ -Glu ^b -Thr ⁶ -Cys ^/ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Tyr ¹⁵ 213 N133 C4:C12 Gln ¹ -Glu ² -Glu ³ -Cys ⁴ -Glu ^b -Glu ⁶ -Cys ^/ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ^{12 -Thr 13} -Gly ¹⁴ -Tyr ¹⁵ 214 N134 C4:C12 Gln ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ^b -Glu ⁶ -Cys ^/ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ^{12 -Thr 13} -Gly ¹⁴ -Tyr ¹⁵ 215

- 32 042294

N135 04:012 6ly ^l4 -1yr ¹⁵ 216 N136 04:012 61n ¹ -61u ² -Asp ³ -Cys ⁴ -61u ^b -61u ⁶ -Cys ^/ -Ile ⁸ -Asn ⁹ -Met 10 -Ala ^{11 -Cys 12} -Thr ¹³ 61y ¹⁴ -Tyr ¹⁵ 217 N137 04:012 611/-611^-611^^3^611^^^^3^116^3^-146 0^^13^^3^-1111:^61y ¹⁴ -Tyr ¹⁵ 218 N138 04:012 61p ¹ -Azr ² -61i ³ -Suz ⁴ -611G-Tug ⁶ -Suz ^/ -11e ⁸ -AzgG-Me0 ¹ ° -A1z ¹¹ -Suz ¹² -T11g ¹³ 61y ¹⁴ -Tyr ¹⁵ 219 N139 04:012 61n ⁱ -Asp ² -Asp ³ -Cys ⁴ -61u ³ -Tyr ⁶ -Cys ^/ -Ile ⁸ -Asn ^y -Met ^iU -A13 ⁱⁱ -0ys ⁱ² -Thr ⁱ³ - 61y ¹⁴ -Tyr ¹⁵ 220 N140 04:012 61n ⁱ -61u ² -Asp ³ -Cys ⁴ -61u ³ -Tyr ^b -Cys ^/ -Ile ⁸ -Asn ^y -Met ^iU -A13 ⁱⁱ -0ys ⁱ² -Thr ⁱ³ 61y ¹⁴ -Tyr ¹⁵ 221 N141 04:012 611/-611/-611/^3^611/-1^^3^116^31/-146 0^^13^^3^-1111:^61y ¹⁴ -Tyr ¹⁵ 222 N142 04:012 61n ¹ -Asp ² -61u ³ -Cys ⁴ -61i/-Ile ⁶ -0ys ^/ -Ile ⁸ -Asn ⁹ -MeO ^lo -Al3 ^ll -Cys ^l2 -1hr ¹³ 6ly ^l4 -1yr ¹⁵ 223 N143 04:012 6ln ⁱ -Asp ² -Asp ³ -Cys ⁴ -6li/-Ile ⁶ -Cys ^/ -Ile ⁸ -Asn ^y -Me0 ^iU -Al3 ⁱⁱ -0ys ⁱ² -1hr ⁱ³ 6ly ^l4 -1yr ¹⁵ 224 N144 04:012 6ln ^l -6lu ² -Asp ³ -Cys ⁴ -6li/-Ile ⁶ -Cys ^/ -Ile ⁸ -Asn ⁹ -Me0 ^l0 -Ala ^ll -Cys ^l2 -1hr ¹³ 6ly ^l4 -1yr ¹⁵ 225 N145 04:012, 07:015 6ln ⁱ -6lu ² -6lu ³ -Cys ⁴ -6lu ³ -1hr ^b -Cys ^/ -Ile ⁸ -Asn ⁹ -MeO ^iU -Al3 ⁱⁱ -Cys ⁱ² -1hr ⁱ³ - 61y ¹⁴ -0ys ¹⁵ -Ser ¹⁶ 226 N146 04:012, 07:015 6ln ⁱ -Asp ² -6lu ³ -Cys ⁴ -6lu ³ -1hr ⁶ -Cys ^/ -Ile ⁸ -Asn ⁹ -Me0 ^iU -Al3 ⁱⁱ -Cys ⁱ² -1hr ⁱ³ - 61y ¹⁴ -0ys ¹⁵ -Ser ¹⁶ 227 N147 04:012, 07:015 61g / -Azr ² -Azr ³ -Cuz ⁴ -611 / -P1g ⁶ -Cuz ^/ -Ile ⁸ -Asn ⁹ -Me0 ^iU -Al3 ⁱⁱ -0ys ⁱ² -1hr ⁱ³ - 61y ¹⁴ -0ys ¹⁵ -Ser ¹⁶ 228 N148 04:012, 07:015 Cln ¹ -61u ² -Asp ³ -Cys ⁴ -61u ^b -1hr ⁶ -Cys ^/ -Ile ⁸ -Asn ⁹ -14e0 ¹⁰ -A13 ¹¹ -Cys ¹² -1hr ¹³ 61y ¹⁴ -0ys ¹⁵ -Ser ¹⁶ 229 N149 04:012, 07:015 61g / -611 / -61u ³ -Suz ⁴ -61i ³ -61u ^b -Suz ^{/ -Ile 8} -Asn ^{9 -Me0 iU -Al3 ii} -0ys ⁱ² -1hr ⁱ³ - 61y ¹⁴ -Cys ¹⁵ -Ser ¹⁶ 230 N150 04:012, 07:015 61r / -Asp ² - 61u ³ -Cys ⁴ -61i / -61u ^b -Cys ^/ -11e ⁸ -Azp ⁹ -Me0 ^1u -A1z ¹¹ -0uz ¹² - 1hr ^{l3 -6ly l4} -0ys ^l5 -Ser 231 N151 04:012, 07:015 61n ¹ -Asp ² -Asp ³ -Cys ⁴ -61i/-61u ⁶ -0ys ^/ -Ile ⁸ -Asn ⁹ -MeO ^lo -Al3 ^ll -Cys ^l2 -1hr ¹³ - 61y ¹⁴ -0ys ¹⁵ -Ser ¹⁶ 232 N152 04:012, 07:015 6ln ^l -6lu ² -Asp ³ -Cys ⁴ -6lu ^b -6lu ⁶ -Cys ^/ -Ile ⁸ -Asn ⁹ -Me0 ^l0 -Al3 ^ll -Cys ^l2 -1hr ¹³ - 61y ¹⁴ -0ys ¹⁵ -Ser ¹⁶ 233 N153 04:012, 07:015 611/-611/-611/^3^611/-^:/^3^116^31/-1460^^13^^3^-1111:^- 61y ¹⁴ -0ys ¹⁵ -Ser ¹⁶ 234 N154 04:012, 07:015 6ln ⁱ -Asp ² -6lu ³ -Cys ⁴ -6lu ³ -1yr ⁶ -Cys ^/ -Ile ⁸ -Asn ⁹ -Me0 ^iU -Al3 ⁱⁱ -0ys ⁱ² -1hr ⁱ³ - 61y ¹⁴ -0ys ¹⁵ -Ser ¹⁶ 235 N155 04:012, 07:015 6ln ^l -Asp ² -Asp ³ -Cys ⁴ -6lu ^b -1yr ⁶ -Cys ^/ -Ile ⁸ -Asn ⁹ -Me0 ^l0 -Al3 ^ll -Cys ^l2 -1hr ¹³ - 61y ¹⁴ -0ys ¹⁵ -Ser ¹⁶ 236 N156 04:012, 07:015 61g / -611 / -Azr ³ -Cuz ⁴ -611 / -1yr ⁶ -Cuz ^/ -11e ⁸ -Azp ⁹ -Me0 ¹⁰ -A1z ¹¹ -Cuz ¹² -1bg ¹³ - 61y ¹⁴ -0ys ¹⁵ -Ser ¹⁶ 237 N157 04:012, 07:015 61g / -611 / -611 / -Cuz ⁴ -611 / -11e ^b -Cuz ^/ -Ile ⁸ -Asn ⁹ -MeO ^iU -Al3 ⁱⁱ -Cys ⁱ² -1hr ⁱ³ - 61y ¹⁴ -0ys ¹⁵ -Ser ¹⁶ 238 N158 04:012, 07:015 6ln ⁱ -Asp ² -6lu ³ -Cys ⁴ -6li/-Ile ⁶ -Cys ^/ -Ile ⁸ -Asn ^y -Me0 ^iU -Al3 ⁱⁱ -0ys ⁱ² -1hr ⁱ³ 61y ¹⁴ -0ys ¹⁵ -Ser ¹⁶ 239 N159 04:012, 07:015 61r/-Asp ² -Asp ³ -Cys ⁴ -61i/-Ile ⁶ -Cys ^/ -Ile ⁸ -Asn ^y -Me0 ^iU -Al3 ⁱⁱ -0ys ⁱ² -1hr ⁱ³ 61y ¹⁴ -Cys ¹⁵ -Ser ¹⁶ 240 N160 04:012, 07:015 6ln ^l -6lu ² -Asp ³ -Cys ⁴ -6li/-Ile ⁶ -Cys ^/ -Ile ⁸ -Asn ⁹ -Me0 ^l0 -Al3 ^ll -Cys ^l2 -1hr ¹³ - 61y ¹⁴ -0ys ¹⁵ -Ser ¹⁶ 241

- 33 042294

Table VII. Peptide ST and analogues

Name Position of disulfide bonds Structure SEQ ID NO Peptide ST SZ:S8, C4:C12, C7: 15 Asn ¹ -Ser ² -Ser ³ -Asn ⁴ -Ser ⁵ -Ser ⁶ -Asn ^{7 -Tyr 8} -Cys 9 -Cys ¹⁰ -Glu ¹¹ -Lys ¹² -Cys ¹³ Cys ¹⁴ -Asn ¹⁵ -Pro ¹⁶ -Ala ¹⁷ - Cys ¹⁸ -Thr ¹⁹ -Gly ²⁰ -Cys ²¹ -Tyr ²² 242 N161 SZ:S8, C4:C12, C7: 15 PEG3-Asn ¹ -Phe ² -Cys ³ -Cys ⁴ -Glu ^{5 -Thr 6 -Cys 7 -Cys} 8 -Asn 9 -Pro ¹⁰ -Ala ¹¹ -Cys ¹² - Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Tyr ¹⁶ - PEG3 243 N162 SZ:S8, C4:C12, C7: 15 PEG3- ^{Asn 1} -Phe ² -Cys ³ -Cys ⁴ -Glu ⁵ -Thr ⁶ -Cys ⁷ -Cys 8 -Asn ⁹ -Pro ¹⁰ -Ala ¹¹ -Cys ¹² - Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Tyr ¹⁶ 244 N163 SZ:S8, C4:C12, C7: 15 Asn ¹ -Phe ² -Cys ³ -Cys ⁴ -Glu ⁵ -Thr ^{6 -Cys 7} -Cys 8 -Asn ⁹ -Pro ^{10 -Ala 11} -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Tyr ¹⁶ -PEG3 245 N164 SZ:S8, C4:C12, C7: 15 Asn ¹ -Phe ² -Cys ³ -Cys ⁴ -Glu ⁵ -Tyr ⁶ -Cys ⁷ -Cys ⁸ -Asn ⁹ -Pro ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Tyr ¹⁶ 246 N165 SZ:S8, C4:C12, C7: 15 dAsn ¹ -Phe ² -Cys ³ -Cys ⁴ -Glu ⁵ -Tyr ^{6 -Cys 7} -Cys 8 -Asn ⁹ -Pro ^{10 -Ala 11} -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dTyr ¹⁶ 247 N166 SZ:S8, C4:C12, C7: 15 Asn ¹ -Phe ² -Cys ³ -Cys ⁴ -Glu ⁵ -Tyr ⁶ -Cys ⁷ -Cys ⁸ -Asn ⁹ -Pro ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dTyr ¹⁶ 248 N167 SZ:S8, C4:C12, C7: 15 dAsn ¹ -Phe ² -Cys ³ -Cys ⁴ -Glu ⁵ -Tyr ⁶ -Cys ⁷ -Cys ⁸ -Asn ⁹ -Pro ^{10 -Ala 11} -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Tyr ¹⁶ 249

1.2. Application methods.

The invention relates to methods for treating or preventing gastrointestinal disorders and increasing gastrointestinal motility in a subject in need thereof by administering an effective amount of a GCC agonist or formulation to the subject. Non-limiting examples of disorders of the gastrointestinal tract that can be treated or prevented by the methods of the invention include irritable bowel syndrome (IBS), non-ulcer dyspepsia associated with H. pylori infection, ulcers, chronic intestinal pseudo-obstruction, functional dyspepsia, colon pseudo-obstruction, duodenogastric reflux, gastroesophageal reflux disease (GERD), ileus (eg, postoperative ileus), gastroparesis, heartburn (high acidity in the GI tract), constipation (eg, constipation associated with the use of drugs such as opioids, anti-osteoarthritis drugs, or drugs anti-osteoporosis agents) postoperative constipation, constipation associated with neuropathic disorders, Crohn's disease and ulcerative colitis.

In one embodiment, the invention relates to methods for treating or preventing gastrointestinal motility disorder, irritable bowel syndrome, functional gastrointestinal disorder, gastroesophageal reflux disease, duodenogastric reflux, functional heartburn, dyspepsia, functional dyspepsia, non-ulcer dyspepsia, gastroparesis, chronic intestinal pseudo-obstruction, pseudo-obstruction of the colon, obesity, congestive heart failure or benign prostatic hyperplasia.

In one embodiment, the invention relates to methods for treating or preventing constipation and/or increasing gastrointestinal motility in a subject in need thereof by administering an effective amount of a GCC agonist or formulation to the subject. Clinically accepted criteria for determining constipation lie in the range of frequency of bowel movements, fecal consistency, and ease of bowel movement. One commonly accepted definition of constipation is fewer than three bowel movements per week. Other definitions include abnormally hard stools or defecation requiring excessive strain (Schiller 2001 Aliment Pharmacol Ther 15:749-763). Constipation may be idiopathic (functional constipation or hypokinetic constipation) or secondary to other causes, including neurological, metabolic, or endocrine disorders. These disorders include diabetes mellitus, hypothyroidism, hyperthyroidism, hypocalcemia, multiple sclerosis, Parkinson's disease, spinal cord injury, neurofibromatosis, autonomic neuropathy,

- 34 042294

Chagas, Hirschsprung disease and cystic fibrosis. Constipation can also result from surgery or be due to the use of drugs such as analgesics (like opioids), antihypertensives, anticonvulsants, antidepressants, anticonvulsants, and antipsychotics.

In various embodiments, the constipation is drug-associated; constipation is associated with a neuropathic disorder; constipation is post-surgical constipation; constipation is associated with a disorder of the gastrointestinal tract; the constipation is idiopathic (functional constipation or hypokinetic constipation); constipation is associated with a neuropathic, metabolic, or endocrine disorder (eg, diabetes mellitus, hypothyroidism, hyperthyroidism, hypocalcemia, multiple sclerosis, Parkinson's disease, spinal cord injury, neurofibromatosis, autonomic neuropathy, Chagas disease, Hirschsprung disease, or cystic fibrosis). Constipation may also result from surgery or be due to the use of drugs such as analgesics (eg, opioids), antihypertensives, anticonvulsants, antidepressants, anticonvulsants, and antipsychotics.

In one embodiment, the invention relates to methods for treating or preventing chronic idiopathic constipation and increasing gastrointestinal motility in a subject in need thereof by administering an effective amount of a GCC agonist or formulation to the subject.

The term treatment, as used herein, refers to the reduction, partial improvement, alleviation or alleviation of at least one clinical symptom associated with the disorder of the gastrointestinal tract being treated. The term prevention refers to inhibiting or delaying the onset or progression of at least one clinical symptom associated with the disorders of the gastrointestinal tract to be prevented. The term effective amount, as used herein, refers to an amount that provides some improvement or advantage to the subject. In certain embodiments, the effective amount is an amount that provides some relief, alleviation and/or reduction of at least one clinical symptom of the gastrointestinal disorder being treated. In other embodiments, the effective amount is an amount that provides some inhibition or delay in the onset or progression of at least one clinical symptom associated with the gastrointestinal disorder to be prevented. Therapeutic effects need not be complete or curative, provided that some benefit is provided to the subject. The term subject preferably refers to a human subject, but may also refer to a non-human primate or other mammal, preferably selected from a mouse, rat, dog, cat, cow, horse, or pig.

The invention also relates to methods for treating gastrointestinal cancer in a subject in need thereof by administering an effective amount of a GCC agonist or formulation to the subject. Non-limiting examples of gastrointestinal cancers that can be treated with the methods of the invention include gastric cancer, esophageal cancer, pancreatic cancer, colorectal cancer, colon cancer, anal cancer, liver cancer, gallbladder cancer, or colon cancer.

The invention also relates to methods for treating lipid metabolism disorders, biliary tract disorders, inflammatory disorders, lung disorders, malignant tumors, cardiac disorders including cardiovascular disorders, eye damage, oral cavity damage, blood disorders, liver disorders, skin damage, prostate disorders. glands, endocrine disorders and obesity.

Lipid metabolism disorders include, but are not limited to, dyslipidemia, hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, sitosterolemia, familial hypercholesterolemia, xanthoma, combined hyperlipidemia, lecithincholesterol acyltransferase deficiency, Tangier disease, abetalipoproteinemia, erectile dysfunction, hepatic steatosis, and hepatitis.

Biliary tract disorders include gallbladder disorders such as, for example, gallstones, cholangitis in gallbladder cancer, or primary sclerosing cholangitis; or disorders of the bile ducts such as, for example, cholecystitis, bile duct cancer, or fascioliasis.

Inflammatory disorders include inflammation of tissues and organs, such as inflammation of the kidneys (eg, nephritis), inflammation of the gastrointestinal system (eg, Crohn's disease and ulcerative colitis); necrotizing enterocolitis (NEC); pancreatic inflammation (eg pancreatitis), pancreatic insufficiency, lung inflammation (eg bronchitis or asthma) or skin inflammation (eg psoriasis, eczema).

- 35 042294

Lung disorders include, for example, chronic obstructive pulmonary disease (COPD) and fibrosis.

Malignant tumor includes carcinogenesis of tissues and organs, including metastasis, such as, for example, a malignant tumor of the gastrointestinal tract, (for example, gastric cancer, esophageal cancer, pancreatic cancer, colorectal cancer, intestinal cancer, anal cancer, liver cancer, cancer gallbladder or colon cancer; lung cancer; thyroid cancer; skin cancer (such as melanoma); oral cancer; urinary tract cancer (such as bladder or kidney cancer); blood cell cancer (such as myeloma). or leukemia) or prostate cancer.

Cardiac disorders include, for example, congestive heart failure, tracheal heart hypertension, high cholesterol or high triglycerides. Cardiovascular disorders include, for example, aneurysm, angina pectoris, atherosclerosis, cerebrovascular accident (stroke), cerebrovascular disease, congestive heart failure, coronary artery disease, myocardial infarction (heart attack) or peripheral vascular disease.

Liver disorders include, for example, cirrhosis and fibrosis. In addition, a GC-C agonist can also be used to facilitate liver regeneration in liver transplant patients. Eye injuries include, for example, increased intraocular pressure, glaucoma, dry eye syndrome, retinal degeneration, damage to the lacrimal glands, or eye inflammation. Skin lesions include, for example, xerosis. Injuries to the oral cavity include, for example, dry mouth (xerostomia), Sjögren's syndrome, gum disease (eg, periodontal disease), or blockage or dysfunction of the salivary gland ducts. Prostate disorders include, for example, benign prostatic hyperplasia (BPH). Endocrine disorders include, for example, diabetes mellitus, hyperthyroidism, hypothyroidism, and cystic fibrosis.

1.2.1. Therapeutically effective doses.

Disorders are treated, prevented, or alleviated by administering to a subject, eg, a mammal, such as a human in need thereof, a therapeutically effective dose of a GCC peptide agonist. The present invention is based in part on unexpected results from human clinical trials showing that the compositions of the invention are therapeutically effective at doses much lower than predicted from animal studies. In one aspect of the invention, the therapeutically effective dose is between 0.01 milligram (mg) and 10 mg per unit dose. The term unit dose refers to a single unit of drug delivery, such as a tablet, capsule, solution, inhalation, controlled release or extended release formulation (eg MMX® technology from Cosmo Pharmaceuticals). In one embodiment, the effective dose is between 0.01 and 9 mg. In another embodiment, the effective dose is between 0.01 and 5 mg. In another embodiment, the effective dose is between 0.01 and 3 mg. In another embodiment, the effective dose is between 0.10 and 5 mg. In another embodiment, the effective dose is between 0.10 and 3 mg. In one embodiment, the unit dose is 0.01, 0.05, 0.1, 0.2, 0.3, 0.5, 1.0, 1.5, 2.0, 2.5, 3 .0, 5 or 10 mg. In one embodiment, the unit dose is 0.3, 1.0, 3.0, 9.0, or 9.5 mg.

GCC agonist peptides may be present in a pharmaceutical composition in unit dosage form, along with one or more pharmaceutically acceptable excipients. The amount of peptide present should be sufficient to provide a beneficial therapeutic effect when administered to a patient. What constitutes a positive therapeutic effect depends on the particular condition being treated and includes any significant improvement readily ascertainable by one of skill in the art.

GCC agonists for use in the methods described above are preferably administered orally. Dosage forms include solutions, suspensions, emulsions, tablets and capsules.

The total daily dose may be administered to the patient in a single dose or in multiple sub-doses. Generally, subdoses may be administered two to six times per day, preferably two to four times per day, and even more preferably two to three times per day. Preferably, a single daily dose is administered.

GCC agonists can be administered either as a single active agent or in combination with one or more additional active agents. In all cases, additional active agents should be administered at a dose that is therapeutically effective, using the current state of the art as a guide. GCC agonists can be administered in a single composition or sequentially with one or more additional active agents. In one embodiment, the GCC agonist is administered in combination with one or more cGMP-dependent phosphodiesterase inhibitors such as sulindac sulfone, zaprinast, motapizone, vardenafil, or sildenafil. In another embodiment, the GCC agonist is administered in combination with one

- 36 042294 him or several chemotherapeutic agents. In another embodiment, the GCC agonist is administered in combination with one or more anti-inflammatory drugs such as steroids or non-steroidal anti-inflammatory drugs (NSAIDS) such as aspirin.

Combination therapy can be achieved by administering two or more agents, for example, a GCC agonist peptide described herein and another compound, each formulated and administered separately, or by administering two or more agents in a single formulation. Other combinations are also included in combination therapy. For example, two agents may be formulated together and administered in combination with a separate formulation containing a third agent. While two or more agents in combination therapy may be administered simultaneously, this is not necessary. For example, administration of the first agent (or combination of agents) may precede administration of the second agent (or combination of agents) by several minutes, hours, days, or weeks. Thus, two or more agents may be administered within minutes of each other, or within 1, 2, 3, 6, 9, 12, 15, 18, or 24 hours of each other, or within 1, 2, 3 , 4, 5, 6, 7, 8, 9, 10, 12, 14 days apart, or within 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks apart. In some cases, even longer intervals are possible. While in many cases it is desirable that two or more agents used in combination therapy be present in the patient's body at the same time, this is not necessary.

The GCC agonist peptides described herein can be combined with phosphodiesterase inhibitors such as sulindac sulfone, zaprinast, sildenafil, vardenafil or tadalfil to further increase cGMP levels in target tissues or organs.

Combination therapy may also include two or more administrations of one or more agents used in combination. For example, if agent X and agent Y are used in combination, they should be administered sequentially in any combination one or more times, for example, in such an order as X-Y-X, X-X-Y, Y-X-Y, Y-Y-X, X-X-Y-Y, etc.

1.2.2. Exemplary Combination Therapies.

The GCC agonist formulations of the invention may be administered alone or in combination with one or more additional therapeutic agents as part of a therapeutic regimen for the treatment or prevention of a disease or disorder of the gastrointestinal tract. In some embodiments, the GCC agonist formulation contains one or more additional drugs. In other embodiments, the GCC agonist is formulated separately from one or more additional drugs. According to this embodiment, the GCC agonist is administered simultaneously, sequentially, or at a different time than one or more additional drugs. In one embodiment, the GCC agonist formulation is administered in combination with one or more additional drugs selected from the group consisting of phosphodiesterase inhibitors, cyclic nucleotides (such as cGMP and cAMP), laxative (such as senna or metamucil), emollient stool, anti-IBD drugs directed against tumor necrosis factor alpha (such as Remicade, Enbrel or Humira), and anti-inflammatory drugs (such as COX-2 inhibitors, sulfasalazine, 5-ASA derivatives and NSAIDs). In certain embodiments, the GCC agonist formulation is administered in combination with an effective dose of a cGMP-specific phosphodiesterase (cGMP-PDE) inhibitor, either simultaneously or sequentially with said GCC agonist. The cGMP-PDE inhibitors include, for example, sulindac-sulfone, zaprinast, motapizone, vardenafil and sildenafil. In another embodiment, the GCC agonist formulation is administered in combination with cyclic nucleotide transporter inhibitors.

Additional examples of therapeutic agents that can be administered in combination with the GCC agonist formulations of the invention are provided in the following sections.

1.2.2.1. Means for the treatment of malignant tumors of the gastrointestinal tract.

The GCC agonist formulations described herein may be used in combination with one or more anticancer agents, including, but not limited to, alkylating agents, epipodophyllotoxins, nitrosoureas, antimetabolites, vinca alkaloids, anthracycline antibiotics, nitrogen mustard agents, and the like. P. Specific anticancer agents include tamoxifen, taxol, etoposide, and 5-fluorouracil. In one embodiment, GCC agonist formulations are used in combination with an antiviral agent or monoclonal antibody.

Non-limiting examples of antitumor agents that can be used in combination with the GCC agonist formulations of the invention for the treatment of colon cancer include antiproliferative agents, DNA modification or repair agents, DNA synthesis inhibitors, DNA/RNA transcription regulators, RNA processing inhibitors, agents agents affecting protein expression, synthesis and stability, agents affecting the localization of proteins or their ability to exert their physiological effects, agents interfering with protein-protein or protein-nucleic acid interactions, agents acting through RNA interference, binding

- 37 042294 receptor molecules of any chemical nature (including small molecule compounds and antibodies), targeted toxins, enzyme activators, enzyme inhibitors, gene regulators, HSP-90 inhibitors, molecules that interfere with microtubules or other components of the cytoskeleton, or with cell adhesion and motility , means for phototherapy and auxiliary therapeutic means.

Representative antiproliferative agents include N-acetyl-O-sphingosine (C.sub.2 ceramide), apigenin, berberine chloride, dichloromethylenediphosphonic acid disodium, aloeemodin, emodin, HA 14-1, N-hexanoyl-O-sphingosine (C .sub.6 ceramide), 7b-hydroxycholesterol, 25hydroxycholesterol, hyperforin, parthenolide and rapamycin. Representative DNA modification and repair agents include afidicolin, bleomycin sulfate, carboplatin, carmustine, chlorambucil, cyclophosphamide monohydrate, ISOPAC.RTM. mitoxantrone dihydrochloride, oxaliplatin and streptozocin.

Representative inhibitors of DNA synthesis include (+-)ametopterin (methotrexate), 3-amino-1,2,4-benzotriazine 1,4-dioxide, aminopterin, cytosine-L-O-arabinofuranoside (Ara-C), cytosine-B hydrochloride -O-arabinofuranoside (Ara-C), 2-fluoroadenine-9-L-O-arabinofuranoside (fludarabine des-phosphate; F-ara-A), 5-fluoro-5'-deoxyuridine, 5-fluorouracil, ganciclovir, hydroxyurea , 6-mercaptopurine and 6-thioguanine.

Representative regulators of DNA/RNA transcription include actinomycin O, daunorubicin hydrochloride, 5,6-dichlorobenzimidazole-1-b-O-ribofuranoside, doxorubicin hydrochloride, homogarringtonin, and idarubicin hydrochloride.

Representative enzyme activators and inhibitors include forskolin, OL aminoglutethimide, apicidin, Bowman-Birk inhibitor, butein, (S)-(+)-camptothecin, curcumin, (-)-deguelin, (-)-depudecin, doxycycline hyclate, etoposide, formestane, fostriyecin sodium, hispidin, 2-imino-1-imidazolidineacetic acid (cyclocreatine), oxamflatine, 4-phenylbutyric acid, roscovitine, sodium valproate, trichostatin A, tyrphostin AG 34, tyrphostin AG 879, urinary trypsin inhibitor fragment, valproic acid (2-propylpentanoic acid) and XK469.

Representative gene regulators include 5-aza-2'-deoxycytidine, 5-azacytidine, chlorcalciferol (vitamin O3), ciglithizone, cyproterone acetate, 15-deoxy-O.sup.12,14-prostaglandin J.sub.2, epitestosterone , flutamide, glycyrrhizic acid ammonium salt (glycyrrhizin), 4-hydroxy tamoxifen, mifepristone, procainamide hydrochloride, raloxifene hydrochloride, all-trans retinol (vitamin A aldehyde), retinoic acid (vitamin A acid), 9-cis-retinoic acid, 13-cis-retinoic acid, retinoic acid p-hydroxyanilide, retinol (vitamin A), tamoxifen, tamoxifen citric acid, tetradecylthioacetic acid, and troglitazone.

Representative HSP-90 inhibitors include 17-(allylamino)-17demethoxygeldanamycin and geldanamycin.

Representative microtubule inhibitors include colchicine, dolastatin 15, nocodazole, taxanes and in particular paclitaxel, podophyllotoxin, rizoxin, vinblastine sulfate salt, vincristine sulfate salt and vindesine sulfate salt and vinorelbine ditartrate salt (navelbin).

Representative phototherapy agents include photoactive porphyrin rings, hypericin, 5-methoxypsoralen, 8-methoxypsoralen, psoralen, and ursodeoxycholic acid.

Representative agents used as ancillary drugs include amifostine, 4-amino-1,8-naphthalimide, brefeldin A, cimetidine, fosfomycin disodium salt, leuprolide (leuprorelin) acetate salt, luteinizing hormone (LH) releasing hormone acetate salt -RH), lectin, papaverine hydrochloride, pifithrin-a, (-)-scopolamine hydrobromide and thapsigargin.

The agents may also be anti-VEGF (vascular endothelial growth factor) agents such as are known in the art. Several antibodies and small molecule compounds that function through VEGF inhibition are currently in clinical trials or have been approved, such as Avastin (bevacizumab), SU5416, SU11248, and BAY 43-9006. The agents may also target growth factor receptors such as the EGF/ErbB family of receptors, e.g. rituxan/mabthera), tyrosine kinases, non-receptor tyrosine kinases, cell series/threonine kinases (including MAP kinases), and various other proteins whose dysregulation contributes to tumorigenesis (eg, small G proteins/Ras families and large/heterotrimeric G proteins). Several antibodies and small molecule compounds targeting these molecules are currently in various stages of development (including those approved for treatment or clinical trials). In a preferred embodiment, the invention relates to a method of treating colon cancer in a subject in need thereof by administering to the subject a GCC agonist formulation in combination with one or more anticancer agents selected from the group consisting of paclitaxel, docetaxel, tamoxifen, vinorelbine, gemcitabine, cisplatin , etoposide, topotecan, irinotecan, ana

- 38 042294 strozol, rituximab, trastuzumab, fludarabine, cyclophosphamide, gentuzumab, carboplatin, interferons and doxorubicin. In a specific embodiment, the anticancer agent is paclitaxel. In a further embodiment, the method further comprises an anticancer agent selected from the group consisting of 5-FU, doxorubicin, vinorelbine, cytoxan, and cisplatin.

1.2.2.2. Treatments for Crohn's disease.

In one embodiment, the GCC agonist formulation of the invention is administered as part of a combination therapy with one or more additional drugs for the treatment of Crohn's disease. Non-limiting examples of one or more additional drugs include sulfasalazine and other mesalamine-containing drugs commonly known as 5-ASA agents such as asacol, dipentum or pentase, or infliximab (Remicade). In certain embodiments, the one or more additional agents are a corticosteroid or an immunosuppressive agent such as 6-mercaptopurine or azathioprine. In another embodiment, the one or more additional agents are an antidiarrheal such as diphenoxylate, loperamide, or codeine.

1.2.2.3. Agents for the treatment of ulcerative colitis.

In one embodiment, the GCC agonist formulation of the invention is administered as part of a combination therapy with one or more additional drugs for the treatment of ulcerative colitis. Drugs used to treat ulcerative colitis overlap with drugs used to treat Crohn's disease. Non-limiting examples of one or more additional drugs that can be used in combination with the GCC agonist formulation of the invention include aminosalicylates (drugs containing 5-aminosalicylic acid (5-ASA)), such as sulfasalazine, olsalazine, mesalamine, and balsalazide. Other drugs that may be used include corticosteroids such as prednisone and hydrocortisone, immunomodulators such as azathioprine, 6-mercaptopurine (6-MP), cytokines, interleukins, and lymphokines, and anti-TNF-alpha agents, including thiazolidinediones or glitazones such as rosiglitazone and pioglitazone. In one embodiment, one or more additional drugs include both cyclosporin A and 6-MP or azathioprine for the treatment of active, severe ulcerative colitis.

1.2.2.4. Means for the treatment of constipation / irritable bowel syndrome.

In one embodiment, the GCC agonist formulation of the invention is administered as part of a combination therapy with one or more additional drugs for the treatment of constipation, such as constipation associated with irritable bowel syndrome. Non-limiting examples of one or more additional drugs include laxatives such as senna, miralax, lactulose, PEG, or calcium polycarbophil), a stool softener (such as mineral oil or colas), bulking agents (such as metamucil or bran), such such as zelnorm (also called tegaserod) and anticholinergic drugs such as bentyl and levsin.

1.2.2.5. Means for the treatment of postoperative ileus.

In one embodiment, the GCC agonist formulation of the invention is administered as part of a combination therapy with one or more additional drugs for the treatment of postoperative ileus.

Non-limiting examples of one or more additional drugs include entereg (alvimopan; formerly called adolor/ADL 8-2698), conivaptan, and related agents described in US6645959.

1.2.2.6. Means against obesity.

In one embodiment, the GCC agonist formulation of the invention is administered as part of a combination therapy with one or more additional drugs for the treatment of obesity. Non-limiting examples of one or more additional drugs include 11β HSD-I (11-beta-hydroxysteroid dehydrogenase type 1) inhibitors such as BVT 3498, BVT 2733, 3-(1-adamantyl)-4-ethyl-5-(ethylthio )-4H-1,2, 4-triazole, 3-(1adamantyl)-5-(3,4,5-trimethoxyphenyl)-4-methyl-4H-1,2,4-triazole, 3-adamantanyl-4, 5,6,7,8,9,10,11,12,3adecahydro-1,2,4-triazolo[4,3-a][11] annulene and compounds described in WO01/90091, WO01/90090, WO01 /90092 and WO02/072084; 5HT antagonists such as those in WO03/037871, WO03/037887 and the like; 5HTIa modulators such as carbidopa, benserazide and the modulators described in US6207699, WO03/031439, and the like; 5HT2c (serotonin 2c receptor) agonists such as BVT933, DPCA37215, IK264, PNU 22394, WAY161503, R-1065, SB 243213 (Glaxo Smith Kline) and YM 348, and agonists described in US3914250, WO00/77010, WO902 , WO02/48124, WO02/10169, WO01/66548, WO02/44152, WO02/51844, WO02/40456 and WO02/40457; 5HT6 receptor modulators such as those in WO03/030901, WO03/035061, WO03/039547, and the like; acylestrogens such as oleoilestrone as described in del Mar-Grasa, M. et al, Obesity Research, 9:202-9 (2001) and Japanese Patent Application No. JP2000256190; anorexigenic bicyclic compounds such as 1426 (Aventis) and 1954 (Aventis) and compounds, opi

- 39 042294 sledges in WO00/18749, WO01/32638, WO01/62746, WO01/62747 and WO03/015769; CB 1 (cannabinoid-1 receptor) antagonists/inverse agonists such as rimonabant (Acomplia; Sanofi), SR-147778 (Sanofi), SR-141716 (Sanofi), BAY 65-2520 (Bayer) and SLV 319 (Solvay) and описанные в патентных публикациях US4973587, US5013837, US5081122, US5112820, US5292736, US5532237, US5624941, US6028084, US6509367, US6509367, WO96/33159, WO97/29079, WO98/31227, WO98/33765, WO98/37061, WO98/41519, WO98/ 43635 WO98/43636 WO99/02499 WO00/10967 WO00/10968 WO01/09120 WO01/58869 WO01/64632 WO01/64633 WO01/64634 WO01/70700 WO03/006007, WO03/007887, WO03/020217, WO03/026647, WO03/026648, WO03/027069, WO03/027076, WO03/027114, WO03/037332, WO03/040107, WO03/086940, WO03/084943 и ЕР658546; CCK-A (chocystokinin-A) agonists such as AR-R 15849, GI 181771 (GSK), JMV-180, A-71378, A-71623 and SR146131 (Sanofi) and described in US5739106; CNTF (ciliary neurotrophic factors) such as GI-181771 (Glaxo-SmithKline), SR1 46131 (Sanofi Synthelabo), butabindide, PD 170292 and PD 149164 (Pfizer); CNTF derivatives such as Axokine® (Regeneron) and described in WO94/09134, WO98/22128 and WO99/43813; dipeptidyl peptidase IV (DP-IV) inhibitors such as isoleucine thiazolidide, valine pyrrolidide, NVP-DPP728, LAF237, P93/01, P 3298, TSL 225 (tryptophyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid; described by Yamada et al, Bioorg. & Med. Chem. Lett. 8 (1998) 1537-1540), TMC-2A/2B/2c, CD26 inhibitors, FE 999011, P9310/K364, VIP 0177, SDZ 274-444, 2 -cyanopyrrolidides and 4-cyanopyrrolidides such as those described in Ashworth et al, Bioorg. &Med. Chem. Lett., Vol. 6, no. 22, pp 1163-1166 and 2745-2748 (1996) and compounds described in patent publications. WO99/38501, WO99/46272, WO99/67279 (Probiodrug), WO99/67278 (Probiodrug), WO99/61431 (Probiodrug), WO02/083128, WO02/062764, WO03/000180, WO03/000181, WO03. /002530, WO03/002531, WO03/002553, WO03/002593, WO03/004498, WO03/004496, WO03/017936, WO03/024942, WO03/024965, WO03/033524, WO08/037327; growth hormone secretagogue receptor agonists/antagonists such as NN703, hexarelin, MK-0677 (Merck), SM-130686, CP-424391 (Pfizer), LY 444,711 (Eli Lilly), L-692,429 and L-163,255, and such as those described in USSN 09/662448, US provisional application 60/203335, US6358951, US2002049196, US2002/022637, WO01/56592 and WO02/32888; H3 (histamine H3) antagonists/inverse agonists such as thioperamide, 3-(1-nimidazol-4-yl)propyl N-(4-pentenyl)carbamate), clobenpropit, iodophenpropit, imoproxifan, GT2394 (Gliatech) and A331440, O-[ 3-(1H-imidazol-4-yl)propanol]carbamates (Kiec-Kononowicz, K. et al., Pharmazie, 55:349-55 (2000)), containing histamine H3 receptor antagonists (Lazewska, D. et al. , Pharmazie, 56:927-32 (2001), benzophenone derivatives and related compounds (Sasse, A. et al., Arch. Pharm. (Weinheim) 334:45-52 (2001)), substituted N-phenyl carbamates (Reidemeister, S. et al., Pharmazie, 55:83-6 (2000)) and proxifan derivatives (Sasse, A. et al., J. Med. Chem. 43:3335-43 (2000)) and histamine H3 receptor modulators, such as those described in WO02/15905, WO03/024928 and WO03/024929, leptin derivatives such as those described in US5552524, US5552523, US5552522, US5521283, WO96/23513, WO96/23514, WO96/23515/23515, WO96 , WO96/23518, WO96/23519 and WO96/23520, leptin, including recombinant human leptin (PEG-O B, Hoffman La Roche) and recombinant human methionyl leptin (Amgen); lipase inhibitors such as tetrahydrolipstatin (orlistat/Xenical®), Triton WR1339, RHC80267, lipstatin, tysaponin, diethylumbelliferyl phosphate, FL-386, WAY-121898, Bay-N-3176, valilactone, esteracin, ebelactone A, ebelactone B and RHC 80267 and described in patent publications WO01/77094, US4598089, US4452813, US5512565, US5391571, US5602151, US4405644, US4189438 and US4242453; lipid metabolism modulators such as butyric acid, erythrodiol, ursolic acid, uvaol, betulinic acid, betulin and the like, and the compounds described in WO03/011267; Mc4r (melanocortin 4 receptor) agonists such as CHIR86036 (Chiron), ME-10142, ME-10145 and HS-131 (Melacure) and described in PCT Publication No. WO99/64002 WO00/74679 WO01/991752 WO01/25192 WO01/52880 WO01/74844 WO01/70708 WO01/70337 WO01/91752 WO02/059095 059117 WO02/06276 WO02/12166 WO02/11715 WO02/12178 WO02/15909 WO02/38544 WO02/068387 WO02/068388 WO02/067869 WO03/009847, WO03/009850, WO03/013509 and WO03/031410; Mc5r (melanocortin 5 receptor) modulators such as those described in WO97/19952, WO00/15826, WO00/15790, US20030092041; melanin concentrating hormone 1 receptor (MCHR) antagonists such as T-226296 (Takeda), SB 568849, SNP-7941 (Synaptic) and described in patent publications WO01/21169, WO01/82925, WO01/87834, WO02/051809, WO02/ 06245 WO02/076929 WO02/076947 WO02/04433 WO02/51809 WO02/083134 WO02/094799 WO03/004027 WO03/13574 WO03/15769 WO03/028640 WO03/033480, JP13226269 and JP1437059; mGluR5 modulators such as those described in WO03/029210, WO03/047581, WO03/048137, WO03/051315, WO03/051833, WO03/053922, WO03/059904, and the like; serotonergics such as fenfluramine (such as Pondimin® (N-ethyl-alpha-methyl-3-(trifluoromethyl)benzenethanamine hydrochloride), Robbins), dexfenfluramine (such as Redux® (N-ethyl-alpha-methyl-3( trifluoromethyl)benzenethanamine), Interneuron) and sibutamine ((Meridia®, Knoll/Reductil™), including racemic mixtures, as well as optically pure isomers (+) and (-) and their pharmaceutically acceptable salts,

- 40 042294 solvents, hydrates, clathrates and prodrugs, including their salts of sibutramine hydrochloride monohydrate and compounds described in US4746680, US4806570, and US5436272, US20020006964, WO01/27068, and WO01/62341; NE (norepinephrine) transport inhibitors such as GW 320659, despiramine, talsupram and nomifensin; NPY 1 antagonists such as BIBP3226, J-115814, BIBO 3304, LY-357897, CP671906, GI-264879A and described in US6001836, WO96/14307, WO01/23387, WO99/51600, WO01/85690, WO90185, WO90185 /85173, and WO01/89528; антагонисты NPY5 (нейропептида Y Y5), такие как 152,804, GW-569180A, GW-594884A, GW-587081X, GW-548118X, FR235208, FR226928, FR240662, FR252384, 1229U91, GI-264879A, CGP71683A, LY-377897, LY- 366377, PD-160170, SR-120562A, SR-120819A, JCF104 и Н409/22, и соединения, описанные в патентных публикациях US6140354, US6191160, US6218408, US6258837, US6313298, US6326375, US6329395, US6335345, US6337332, US6329395, US6340683, ЕР01010691 , EP-01044970, WO97/19682, WO97/20820, WO97/20821, WO97/20822, WO97/20823,

WO98/27063, WO00/107409, WO00/185714, WO00/185730, WO00/64880, WO00/68197, WO00/69849,

WO/0113917, WO01/09120, WO01/14376, WO01/85714, WO01/85730, WO01/07409, WO01/02379,

WO01/23388, WO01/23389, W00 1/44201, WO01/62737, WO01/62738, WO01/09120, WO02/20488,

WO02/22592, WO02/48152, WO02/49648, WO02/051806, WO02/094789, WO03/009845, WO03/014083, WO03/022849, WO03/028726 and Norman et al, J. Med. Chem. 43:4288-4312 (2000); opioid antagonists such as nalmefene (REVEX®), 3-methoxynaltrexone, methylnaltrexone, naloxone and naltrexone (eg PT901; Pain Therapeutics, Inc.) and described in US20050004155 and WO00/21509; orexin antagonists such as SB-334867-A and described in patent publications WO01/96302, WO01/68609, WO02/44172, WO02/51232, WO02/51838, WO02/089800, WO02/090355, WO03/023561, WO091 and WO03/037847;

PDE inhibitors (e.g. compounds that slow down the degradation of cyclic AMP (cAMP) and/or cyclic GMP (cGMP) by inhibiting phosphodiesterases, which can lead to a relative increase in intracellular concentrations of cAMP and cGMP; possible PDE inhibitors are primarily compounds listed as members the class consisting of PDE3 inhibitors, the class consisting of PDE4 inhibitors and/or the class consisting of PDE5 inhibitors, in particular substances which may be referred to as mixed PDE3/4 inhibitors or mixed PDE3/4/5 inhibitors) such as described in patent publications DE1470341, DE2108438, DE2123328, DE2305339, DE2305575, DE2315801, DE2402908, DE2413935, DE2451417,

DE2459090, DE2646469, DE2727481, DE2825048, DE2837161, DE2845220, DE2847621, DE2934747,

De3021792, DE3038166, DE3044568, ER000718, ER0008408, ER0010759, ER0059948, ER0075436, ER0096517, EP0112987, ER016948, EP0150937, ER0158380, FIRS

EP0199127, EP0220044, EP0247725, EP0258191, EP0272910, EP0272914, EP0294647, EP0300726,

EP0335386, EP0357788, EP0389282, EP0406958, EP0426180, EP0428302, EP0435811, EP0470805,

EP0482208, EP0490823, EP0506194, EP0511865, EP0527117, EP0626939, EP0664289, EP0671389,

EP0685474, EP0685475, EP0685479, JP92234389, JP94329652, JP95010875, US4963561, US5141931,

WO9117991, WO9200968, WO9212961, WO9307146, WO9315044, WO9315045, WO9318024, WO9319068, WO9319720, WO9319747, WO9319749, WO9319751, WO9325517, WO9402465, WO9406423, WO9412461, WO9420455, WO9422852, WO9425437, WO9427947, WO9500516, WO9501980, WO9503794, WO9504045, WO9504046, WO9505386, WO9508534, WO9509623, WO9509624, WO9509627, WO9509836, WO9514667, WO9514680, WO9514681, WO9517392, WO9517399, WO9519362, WO9522520, WO9524381, WO9527692, WO9528926, WO9535281, WO9535282, WO9600218, WO9601825, WO9602541, WO9611917, DE3142982, DE1116676, DE2162096, ЕР0293063, ЕР0463756, ЕР0482208, ЕР0579496, ЕР0667345 US6331543, US20050004222 (включая описанные в формулах I-XIII и параграфах 37-39, 85-0545 и 557-577), WO9307124, ЕР0163965, ЕР0393500, ЕР0510562, ЕР0553174, WO9501338 и WO9603399, а also PDE5 inhibitors (such as RX-RA-69, SCH-51866, KT-734, vesrinone, zaprinast, SKF-96231, ER-21355, BF/GP-385, NM-702 and sildenafil (Viagra™)), inhibitors PDE4 (such as etazolate, ICI63197, RP73401, imazolidi non (RO-20-1724), MEM 1414 (R1533/R1500; Pharmacia Roche), Denbufillin, Rolipram, Oxagrelate, Nitraquasone, Y-590, DH-6471, SKF-94120, Motapizone, Lixazinone, Indolidan, Olprinon, Atizoram, KS-506-G, Dipamfillin, BMY-43351, Atizoram, Arophyllin, filaminast, PDB-093, UCB-29646, CDP-840, SKF-107806, piclamylast, RS-17597, RS-25344-000, SB-207499, tibenelast, SB-210667, SB211572, SB-211600, SB-212066, SB-212179, GW-3600, CDP-840, mopidamol, anagrelide, ibudilast, amrinone, pimobendan, cilostazol, quasinon and N-(3,5-dichloropyrid-4-yl)-3-cyclopropylmethoxy-4difluoromethoxybenzamide, PDE3 inhibitors (such as ICI153, 100, bemorandan (RWJ 22867), MCI-154, UD-CG 212, sulmazol, ampizone, cilostamide, carbaseran, pyroximone, imazodane, CI-930, siquazodan, adibendan, saterinone, SKF-95654, SDZ-MKS- 492, 349-U-85, emoradan, EMD-53998, EMD-57033, NSP306, NSP-307, revisinone, NM-702, WIN-62582 and WIN-63291, enoximone and milrinone, PDE3/4 inhibitors (such as benafenthrin , trequinsin, ORG-30029, zardaverine, L-686398, SDZ-ISQ-844, ORG-20241, EMD-54622 and tola fentrin) and other PDE inhibitors (such as vinpocetine, papaverine, enprophylline, cilomilast, phenoximon, pentoxifylline, roflumilast, tadalafil (Cialis®), theophylline, and vardenafil (Levitra®); Neuropeptide Y2 (NPY2) agonists include, but are not limited to: YY polypeptide and fragments and variants thereof (e.g., YY3-36 (PYY3-36) (N. Engl. J. Med.

- 41 042294

349:941, 2003; IKPEAPGE DASPEELNRY YASLRHYLNL VTRQRY (SEQ ID NO:XXX)), and PYY agonists such as those described in WO02/47712, WO03/026591, WO03/057235 and WO03/027637; serotonin reuptake inhibitors such as paroxetine, fluoxetine (Prozac™), fluvoxamine, sertraline, citalopram and imipramine and are described in US6162805, US6365633, WO03/00663, WO01/27060, and WO01/162341; thyroid hormone β agonists such as KB-2611 (KaroBioBMS) and those described in WO02/15845, WO97/21993, WO99/00353, GB98/284425, US Provisional Application No. 60/183223, and Japanese Patent Application No. JP2000256190; UCP-I (uncoupling protein-1), 2, or 3 activators such as phytanic acid, 4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl -2-naphthalenyl)-1propenyl]benzoic acid (TTNPB), retinoic acid and described in WO99/00123; β3 (beta-adrenergic receptor 3) agonists such as AJ9677/TAK677 (Dainippon/Takeda), L750355 (Merck), CP331648 (Pfizer), CL-316,243, SB 418790, BRL-37344, L-796568, BMS-196085, BRL-35135A, CGP12177A, BTA-243, GW 427353, Trekadrin, Zeneca D7114, N-5984 (Nisshin Kyorin), LY-377604 (Lilly), SR 59119A and those described in US5541204, US5770615, US5491134, US5778134,5 US57768083 US 5451677, WO94/18161, WO95/29159, WO97/46556, WO98/04526 and WO98/32753, WO01/74782, WO02/32897, WO03/014113, WO03/016276, WO03/016307, WO94307, WO94307, WO08 WO03/037881; noradrenergics including, but not limited to, diethylpropion (such as Tenuate® (1-propanone, 2-(diethylamino)-1-phenyl hydrochloride), Merrell), dextroamphetamine (also known as dextroamphetamine sulfate, dexamphetamine, dexedrine, dexampex, ferndex, oxides II, robes, spancap #1), mazindol ((or 5-(p-chlorophenyl)-2,5-dihydro-3H-imidazo[2,1-a]isoindol-5-ol) such as Sanorex®, Novartis or Mazanor®, Wyeth Ayerst), phenylpropanolamine (or alpha-(1-aminoethyl)benzenemethanol hydrochloride), phentermine ((or 3-[[4,5-dihydro-1H-imidazol-2yl)ethyl monohydrochloride](4-methylphenyl )amino]phenol), such as Adipex-P®, Lemmon, FASTIN®, Smith-Kline Beecham and Ionamin®, Medeva), phendimetrazine ((or L-(+)-tartrate (2S,3S)-3,4- dimethyl-2-phenylmorpholine (1:1)), such as Metra® (Forest), Plegine® (Wyeth-Ayerst), Prelu-2® (Boehringer Ingelheim), and Statobex® (Lemmon), fendamine tartrate (such as Thephorin ® (L-(+)-2,3,4,9-tetrahydro-2-methyl-9-phenyl-1Nindenol[2,1-e]pyridine tartrate (1 :1)), Hoffmann-LaRoche), methamphetamine (such as Desoxyn®, Abbot ((S)-N, (alpha)-dimethylbenzenethanamine hydrochloride)), and phendimetrazine tartrate (such as Bontril® sustained release capsules, amarin (tartrate 3,4-dimethyl-2-phenylmorpholine); boost regulators/inducers of fatty acid oxidation, Famoxin® (Genset); monamine oxidase inhibitors including, but not limited to, befloxatone, moclobemide, brofaromine, fenoxatin, esupron, befol, toloxatone, pyrlindole, amiflamin, sercloremin, basinaprine, lazabemide, milacemide, caroxazone, and other specific compounds as described in WO01/12176; and other anti-obesity agents such as 5HT-2 agonists, ACC (acetyl-CoA carboxylase) inhibitors such as those described in WO03/072197, alpha-lipoic acid (alpha-LA), AOD9604, appetite suppressants such as in WO03 /40107, ATL-962 (Alizyme PLC), benzocaine, benzphetamine hydrochloride (Didrex), kelp (focus vesiculosus), BRS3 (subtype 3 bombesin receptor) agonists, bupropion, caffeine, CCK agonists, chitosan, chromium, conjugated linoleic acid, agonists corticotropin-releasing hormone, dehydroepiandrosterone, DGAT1 (diacylglycerol acyltransferase 1) inhibitors, DGAT2 (diacylglycerol acyltransferase 2) inhibitors, dicarboxyl transporter inhibitors, ephedra, exendin-4 (glp-1 inhibitor), FAS (fatty acid synthase) inhibitors (such as cerulenin and C75 ), fat resorption inhibitors (such as in WO03/053451, etc.), fatty acid transporter inhibitors, natural water-soluble fibers (such as psyllium, psyllium, guar, oats, pectin), galanin antagonists, ha lega (goat's rue, French lilac), garcinia cambogia, dubrovnik (teucri um chamaedrys), anti-ghrelin antibodies and ghrelin antagonists (such as those described in WO01/87335 and WO02/08250), polypeptide hormones and their variants affecting secretion from islets cells, such as hormones from the secretin/gastric inhibitory polypeptide (GIP)/vasoactive gut polypeptide (VIP)/pituitary adenylyl cyclase activating polypeptide (PACAP)/glucagon-like polypeptide II (GLP-Π)/glycentin/glucagon gene family and/or from the gene family adrenomedullin/amylian/calcitonin-related polypeptide (CGRP) GLP-1, including GLP-l(7- 34), GLP-1(7-35), GLP-1(7-36), or GlP-1(7-37) in their C-terminally carboxylated or amidated forms, or in the form of modified GLP-1 polypeptides and modifications thereof , including those described in paragraphs 17-44 of US20050 130891 and use derivatives derived from GLP-1-(7-34)COOH and the corresponding acid amide having the following general formula: R-NH-HAEGTFTSDVSYLEGQAAKEFIAWLVK-CONH ₂ where R=H or an organic compound having 1 to 10 carbon atoms. Preferably R is a carboxylic acid residue. Particularly preferred are the following carboxylic acid residues: formyl, acetyl, propionyl, isopropionyl, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl) and glp-1 (glucagon-like polypeptide-1), glucocorticoid antagonists , glucose transporter inhibitors, growth hormone secretagogues (such as disclosed and specifically described in US5536716), interleukin-6 (IL-6) and its modulators (as in WO03/057237 and the like), L-carnitine, agonists

- 42 042294

Mc3r (melanocortin 3 receptor), MCH2R (melanin concentrating hormone 2R) agonist/antagonists, melanin concentrating hormone antagonists, melanocortin agonists (such as melanotan II or those described in WO99/64002 and WO00/74679), nomame herba, phosphate transporter inhibitors , herbal drug 57 (CP 644,673), pyruvate, SCD-I (stearoyl-CoA desatase-1) inhibitors, T71 (Tularik, Inc., Boulder CO), topiramate (Topimax®, described as an anticonvulsant that, like shown to increase weight loss), transcription factor modulators (such as those described in WO03/026576), β-hydroxysteroid dehydrogenase-1 (β-HSD-I) inhibitors, β-hydroxy-β-methylbutyrate, p57 (Pfizer), zonisamide (Zonegran ™ is described as an antiepileptic agent that has been shown to be capable of causing weight loss) and those described in US20030119428, paragraphs 20-26.

1.2.2.7. Phosphodiesterase inhibitors.

In specific embodiments, the combination therapy regimen comprises the administration of one or more phosphodiesterase (PDE) inhibitors. PDE inhibitors slow down the degradation of cyclic AMP (cAMP) and/or cyclic GMP (cGMP) through the inhibition of phosphodiesterases, which can lead to a relative increase in the intracellular concentration of cAMP and/or cGMP. Non-limiting examples of PDE inhibitors that can be used in combination with the GCC agonists of the invention include PDE3 inhibitors, PDE4 inhibitors and/or PDE5 inhibitors, in particular substances that can be referred to as PDE3/4 mixed-type inhibitors or PDE3 mixed-type inhibitors. /4/5. Non-limiting examples of such PDE inhibitors are described in the following patent applications and patents:

DE2646469, DE2727481, DE2825048, DE2837161, DE2845220, DE2847621, DE2934747, DE3021792,

DE3038166, DE3044568, EP000718, EP0008408, EP0010759, EP0059948, EP0075436, EP0096517,

EP0112987, EP0116948, EP0150937, EP0158380, EP0161632, EP0161918, EP0167121, EP0199127,

EP0220044, EP0247725, EP0258191, EP0272910, EP0272914, EP0294647, EP0300726, EP0335386,

EP0357788, EP0389282, EP0406958, EP0426180, EP0428302, EP0435811, EP0470805, EP0482208,

EP0490823, EP0506194, EP0511865, EP0527117, EP0626939, EP0664289, EP0671389, EP0685474,

EP0685475, EP0685479, JP92234389, JP94329652, JP95010875, US Pat. 4963561, 5141931,

WO9117991, WO9200968, WO9212961, WO9307146, WO9315044, WO9315045, WO9318024, WO9319068, WO9319720, WO9319747, WO9319749, WO9319751, WO9325517, WO9402465, WO9406423, WO9412461, WO9420455, WO9422852, WO9425437, WO9427947, WO9500516, WO9501980, WO9503794, WO9504045, WO9504046, WO9505386, WO9508534, WO9509623, WO9509624, WO9509627, WO9509836, WO9514667, WO9514680, WO9514681, WO9517392, WO9517399, WO9519362, WO9522520, WO9524381, WO9527692, WO9528926, WO9535281, WO9535282, WO9600218, WO9601825, WO9602541, WO9611917, DE3142982, DE1116676, DE2162096, ЕР0293063, ЕР0463756, ЕР0482208, ЕР0579496, ЕР0667345 US6331543, US20050004222 (включая описанные в формулах I-XIII и параграфах 37-39, 85-0545 и 557-577) и WO9307124, ЕР0163965, ЕР0393500, ЕР0510562, ЕР0553174, WO9501338 и WO9603399. Examples of PDE5 inhibitors that may be mentioned are RX-RA-69, SCH-51866, KT734, vesnarinone, zaprinast, SKF-96231, ER-21355, BF/GP-385, NM-702, and sildenafil (Viagra® ). PDE4 inhibitors that may be mentioned by way of example are RO-20-1724, MEM 1414 (R1533/R1500; Pharmacia Roche), denbuphylline, rolipram, oxagrelate, nitraquasone, Y-590, DH-6471, SKF-94120, motapizone , lixazinone, indolidan, olprinon, atizoram, KS-50 6-G, DIPAMFYLLINE, BMY43351, atizoram, arophyllin, filaminast, PDB-093, UCB-29646, CDP-840, SKF-107806, piclamylast, RS-17597, RS- 25344-000, SB-207499, Tibenelast, SB-210667, SB-211572, SB-211600, SB-212066, SB212179, GW-3600, CDP-840, Mopidamol, Anagrelide, Ibudilast, Amrinone, Pimobendan, Cilostazol, Quasinon and N-(3,5-dichloropyrid-4-yl)-3-cyclopropylmethoxy-4-difluoromethoxybenzamide. PDE3 inhibitors that can be mentioned by way of example are sulmazol, ampisone, cilostamide, carbaseran, piroximon, imazodane, CI-930, siquazodan, adibendan, saterinone, SKF-95654, SDZ-MKS492, 349-U-85, emoradan, EMD-53998, EMD-57033, NSP-306, NSP-307, REVIZINONE, NM-702, WIN62582 and WIN-63291, enoximone and milrinone. Examples of PDE3/4 inhibitors that may be mentioned are benafenthrin, trequinsin, ORG-30029, zardaverine, L-686398, SDZ-ISQ844, ORG-20241, EMD-54622, and tolafenthrin. Other PDE inhibitors include: cilomilast, pentoxifylline, roflumilast, tadalafil (Cialis®), theophylline and vardenafil (Levitra®), zaprinast (a PDE5-specific GCC agonist).

1.2.2.8. analgesics.

In certain embodiments, the combination therapy regimen comprises the administration of one or more analgesics, for example, an analgesic compound or an analgesic polypeptide. In some embodiments, the GCC agonist formulation is administered simultaneously or sequentially with one or more analgesics. In other embodiments, the GCC agonist is covalently linked to or linked to an analgesic to form a therapeutic conjugate. Non-limiting examples of analgesics that can be used include calcium channel blockers, 5HT receptor antagonists (e.g., 5HT3, 5HT4, and 5HT1 receptor antagonists), opioid receptor agonists (loperamide, fedotosine, and fentanyl), NK1 receptor antagonists, CCK receptor agonists (eg, loxiglumide), NK1 receptor antagonists, NK3 receptor antagonists, norepinephrine-serotonin reuptake inhibitors (NSRIs), vanilloid and cannabinoid receptor agonists, and sialorphin. Additional examples of analgesics of various classes are known in the art.

In one embodiment, the analgesic is an analgesic polypeptide selected from the group consisting of sialorphin-related polypeptides, including those containing the amino acid sequence QHNPR (SEQ ID NO: 239), including: VQHNPR (SEQ ID NO: 240); VRQHNPR (SEQ ID NO: 241); VRGQHNPR (SEQ ID NO: 242); VRGPQHNPR (SEQ ID NO: 243); VRGPRQHNPR (SEQ ID NO: 244); VRGPRRQHNPR (SEQ ID NO: 245); and RQHNPR (SEQ ID NO: 246). Sialorphin-related polypeptides bind neprilysin and inhibit neprilysin-mediated degradation of substance P and Met-enkephalin. Thus, compounds or polypeptides that are inhibitors of neprilysin are useful analgesics that can be administered with the GCC agonists described herein or covalently linked to a GCC agonist to produce a therapeutic conjugate. Sialorfin and related polypeptides are described in US Pat. No. 6,589,750; US20030078200 A1; and WO02/051435 A2.

In another embodiment, the GCC agonist formulation of the invention is administered as part of a combination therapy regimen with an opioid receptor antagonist or agonist. In one embodiment, the GCC agonist and the opioid receptor antagonist or agonist are linked via a covalent bond. Non-limiting examples of opioid receptor antagonists include naloxone, naltrexone, methylnalosone, nalmefene, cypridim, beta-funaltrexamine, naloxonazine, naltrindol, norbinaltorfimine, enkephalin pentapeptide (HOE825; Tyr-D-Lys-Gly-Phe-Lhomoserine), trimebutine, vasoactive intestinal polypeptide, gastrin, glucagons. Non-limiting examples of opioid receptor agonists include fedotosin, asimadoline and ketocyclazocine, compounds described in WO03/097051 and WO05/007626, morphine, diphenyloxylate, frakefamide (H-TyrD-Ala-Phe(F)-Phe-NH2; WO01/019849 A1) and loperamide.

Additional non-limiting examples of analgesics that can be used in combination therapy with GCC agonist formulations of the invention include Tyr-Arg dipeptide (kyotorphin); a chromogranin-derived polypeptide (CgA 47-66; See, for example, Ghia et al. 2004 Regulatory polypeptides 119:199); CCK receptor agonists such as cerulein; conotoxin polypeptides; peptide analogs of thymulin (French application 2830451); CCK receptor antagonists (CCKa or CCKb), including loxiglumide and dexloxiglumide (R-isomer of loxiglumide) (WO 88/05774); 5-HT4 agonists such as tegaserod (Zelnorm®), mosapride, metoclopramide, zacopride, cisapride, renzapride, benzimidazolone derivatives such as BIMU 1 and BIMU 8 and lirexapride; блокаторы кальциевых каналов, такие как зиконотид и родственные соединения, описанные, например, в ЕР625162В1, US5364842, US5587454, US5824645, US5859186, US5994305, US6087,091, US6136786, WO93/13128 A1, EP1336409 A1, EP835126 A1, EP835126 B1, US5795864, US5891849, US6054429, WO97/01351A1; NK-I receptor antagonists such as aprepitant (Merck & Co Inc.), wofopitant, eslopitant (Pfizer, Inc.), R673 (Hoffmann-La Roche Ltd), SR-48968 (Sanofi Synthelabo), CP-122,721 (Pfizer, Inc. .), GW679769 (Glaxo Smith Kline), TAK-637 (Takeda/Abbot), SR-14033, and related compounds described in e.g. in Giardina et al 2003. Drugs 6:758); NK-2 receptor antagonists such as non-padutant (Menarini Ricerche SpA), saredutant (Sanofi-Synthelabo), GW597599 (Glaxo Smith Kline), SR-144190 (Sanofi-Synthelabo) and UK-290795 (Pfizer Inc); NK3 receptor antagonists such as osanetant (SR-142801; Sanofi-Synthelabo), SSR241586, talnetant and related compounds, as described for example in WO02/094187 A2, EP 876347 A1, WO97/21680 A1, US6277862, WO98/1 1090, WO95/28418, WO97/19927 and Boden et al. (J Med Chem. 39:1664-75, 1996); norepinephrine-serotonin reuptake inhibitors (NSRIs) such as milnacipran and related compounds described in WO03/077897; and vanilloid receptor antagonists such as arvanil and related compounds described in WO01/64212 A1.

In addition to sialorphin-related polypeptides, analgesic polypeptides include: AspPhe, endomorphin-1, endomorphin-2, nocystatin, dalargin, lupron, ziconotide, and substance P.

1.2.2.9. Insulin and insulin modulating agents.

The GCC agonist peptides described herein can be used in combination therapy with insulin and related compounds, including primate, rodent or rabbit insulin, including biologically active variants thereof, including allelic variants, more preferably human insulin available in recombinant form. Human insulin sources include pharmaceutically acceptable and sterile formulations such as those available from Eli Lilly (Indianapolis, Ind. 46285) as H MKMulin™ (human insulin rDNA source). See THE PHYSICIAN'S DESK REFERENCE, 55.sup.th Ed. (2001) Medical Economics, Thomson Healthcare (which describes other useful types of human insulin).

- 44 042294

The GCC peptides described herein can also be used in combination treatment with agents capable of enhancing the effects or increasing insulin levels in a subject after administration, such as glipizide and/or rosiglitazone. The polypeptides and agonists described herein can be used in combination therapy with SYMLIN® (pramlintide acetate) and Exenatide® (synthetic exendin-4; 39 aa polypeptide).

1.2.2.10. Antihypertensive agents.

The GCC agonist peptides described herein can be used in combination therapy with an antihypertensive agent, including, but not limited to: (1) diuretics such as thiazides, including chlorthalidone, chlorthiazide, dichlorophenamide, hydroflumethiazide, indapamide, polythiazide, and hydrochlorothiazide; loop diuretics such as bumetanide, ethacrynic acid, furosemide and torsemide; potassium-sparing diuretics such as amiloride and triamterene; carbonic anhydrase inhibitors, osmotic agents (such as glycerol), and aldosterone antagonists such as spironolactone, epirenone, and the like; (2) beta-adrenergic blockers such as acebutolol, atenolol, betaxolol, bevantolol, bisoprolol, bopindolol, carteolol, carvedilol, celiprolol, esmolol, indenolol, metaprolol, nadolol, nebivolol, pentbutolol, pindolol, propanolol, sotalol, tertatolol, tilizolol, and timolol, etc.; (3) calcium channel blockers such as amlodipine, aranidipine, azelnidipine, barnidipine, benidipine, bepridil, cinaldipine, clevidipine, diltiazem, efonidipine, felodipine, gallopamil, isradipine, lacidipine, lemildipine, lercanidipine, nicardipine, nifedipine, nilvadipine, nisolodipine, nizoldipine , nitrendipine, manidipine, pranidipine and verapamil, etc.; (4) angiotensin converting enzyme (ACE) inhibitors such as benazepril; captopril; cerapril; cilazapril; delapril; enalapril; enalopril; fosinopril; imidapril; lisinopril; losinopril; moexipril; quinapril; quinapril; ramipril; perindopril; perindropril; quanipril; spirapril; tenocapril; trandolapril and zofenopril, etc.; (5) neutral endopeptidase inhibitors such as omapatrilat, cadoxatril and ecadotril, phosidotril, sampatrilat, AVE7688, ER4030, and the like; (6) endothelin antagonists such as tesosentan, A308165 and YM62899 and the like; (7) vasodilators such as hydralazine, clonidine, minoxidil, and nicotinyl alcohol, and the like; (8) angiotensin II receptor antagonists such as aprosartan, candesartan, eprosartan, irbesartan, losartan, olmesartan, pratosartan, tasosartan, telmisartan, valsartan and EXP-3137, FI6828K and RNH6270, etc.; (9) α/β-adrenergic blockers such as nipradiol, arotinolol and amosulalol, etc.; (10) alpha 1 blockers such as terazosin, urapidil, prazosin, tamsulosin, bunazocin, trimazocin, doxazosin, naftopidil, indoramin, WHP 164, and XENOlO, etc.; (11) alpha 2 agonists such as lofexidine, thiamenidine, moxonidine, rilmenidine and quanobenz and the like; (12) aldosterone inhibitors and the like; and (13) angiopoietin-2 binding agents such as those described in WO03/030833. Specific antihypertensive agents that may be used in combination with the polypeptides and agonists described herein include, but are not limited to: diuretics such as thiazides (e.g., chlorthalidone, cyclothiazide (CAS RN 2259-96-3), chlorthiazide (CAS RN 72956-093, which can be prepared as described in US2809194), dichlorophenamide, hydroflumethiazide, indapamide, polythiazide, bendroflumethazide, meticlotazide, polythiazide, trichlormethazide, chlorthalidone, indapamide, metolazone, quinetasone, althiazide (CAS RN 5588-16-9 , which can be obtained as described in British Patent No. 902658), benzthiazide (CAS RN 91-33-8, which can be obtained as described in US3108097), buthiazide (which can be obtained as described in British Patent No. 861367) , and hydrochlorothiazide), loop diuretics (eg, bumetanide, ethacrynic acid, furosemide, and torasemide), potassium-sparing diuretics (eg, amiloride, and triamterene (CAS number 396-01-0)), and aldos antagonists sterone (eg, spironolactone (CAS No. 52-01-7), epirenone, etc.); β-adrenergic blockers such as amiodarone (cordarone, paceron), bunolol hydrochloride (CAS RN 31969-05-8, Parke-Davis), acebutolol (±N-[3-acetyl-4-[2-hydroxy-3-[(1methylethyl )amino]propoxy]phenyl]butanamide or (±)-3'-acetyl-4'-[2-hydroxy-3(isopropylamino)propoxy]butyranilide), acebutolol hydrochloride (e.g. Sectral®, Wyeth-Ayerst), hydrochloride alprenolol (CAS RN 13707-88-5 see Dutch Patent Application No. 6605692), atenolol (e.g. Tenormin®, AstraZeneca), carteolol hydrochloride (e.g. Cartrol® Filmtab®, Abbott), celiprolol hydrochloride (CAS RN 57470-78 -7, see also US4034009), cetamolol hydrochloride (CAS RN 77590-95-5, see also US4059622), labetalol hydrochloride (e.g. Normodyne®, Schering), esmolol hydrochloride (e.g. Brevibloc®, Baxter), levobetaxolol hydrochloride (eg, Betaxon™ ophthalmic suspension, Alcon), levobunolol hydrochloride (eg, Betagan® Liquifilm® with C CAP® Compliance Cap, Allergan), nadolol (eg, nadolol, Mylan), practolol ( CAS RN 6673-35-4, see also US3408387), propranolol hydrochloride (CAS RN 318-98-9), sotalol hydrochloride (e.g. Betapace AF™, Berlex), timolol ((S)-1-[( 1,1-dimethylethyl)amino]-3-[[4-4(4-morpholinyl)-1,2,5thiadiazol-3-yl]oxy]-2-propanol, CAS RN 91524-16-2), maleate salt (S)-1-[(1,1-dimethylethyl)amino]3-[[4-(4-morpholinyl)-1,2,5-thiadiazol-3-yl]oxy]-2-propanol-(Z) -2-butenedioate timolol (1:1), CAS RN 26921-17-5), bisoprolol ((±)-1-[4-[[2-(1-methylethoxy)ethoxy]-methyl]phenoxy]-3- [(1-methylethyl)amino]2-propanol, CAS RN 66722-44-9), bisoprolol fumarate (such as (E)-2-butenedioate (±)-1-[4-[[2-(1

- 45 042294 methylethoxy)ethoxy]methyl]phenoxy]-3-[(1-methylethyl)amino]-2-propanol (2:1) (salt), e.g. Zebeta™, Lederle Cons umeg), nebivalol (α a' -[imino-bis(methylene)]bis[6-fluoro-3,4-dihydro-2H-1benzopyran-2-methanol, CAS RN 99200-09-6 see also US Patent No. 4654362), cycloprolol hydrochloride, such as 1-[4-[2-(cyclopropylmethoxy)ethoxy]phenoxy]-3-[1-methylethyl)amino]2-propanol hydrochloride, A.A.S. RN 63686-79-3), dexpropranolol hydrochloride (1-[1-methylethyl)amino]-3-(1-naphthalenyloxy)-2-propanol hydrochloride (CAS RN 13071-11-9), diacetolol hydrochloride (N-[ 3-acetyl-4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy][phenyl]acetamide, CAS RN 6979604-9), dilevalol hydrochloride (2-hydroxy-5-[1-hydroxy- 2-[1-methyl-3phenylpropyl)amino]ethyl]benzamide, CAS RN 75659-08-4), exaprolol hydrochloride (1-(2cyclohexylphenoxy)-3-[(1-methylethyl)amino]-2-propanol hydrochloride, CAS RN 59333-90-3), flestolol sulfate ((+)-sulfate 2-fluoro-,3-[[2-[aminocarbonyl)amino]dimethylethyl]amino]-2-hydroxypropyl benzoic acid ester (1:1) ( salt), CAS RN 88844-73-9; metalol hydrochloride (N-[4-[1-hydroxy-2-(methylamino)propyl]phenyl]-methanesulfonamide monohydrochloride, CAS RN 7701-65-7), metoprolol, 1-[4-(2-methoxyethyl)phenoxy]- 3-[1-methylethyl)amino]-2-propanol; CAS RN 37350-58-6), metoprolol tartrate (such as 1-[4-(2-methoxyethyl)phenoxy]-3-[(1-methylethyl)amino]-2-propanol, such as Lopressor®, Novartis), pamatolol sulfate ((±) [2-[4-[2-hydroxy-3-[(1methylethyl)amino]propoxy]phenyl]ethyl]carbamic acid methyl ester sulfate (salt) (2:1), CAS RN 59954 -01-7), pentbutolol sulfate ((S)-1-(2-cyclopentylphenoxy)-3-[1,1-dimethylethyl)amino]-2-propanol sulfate (2:1) (salt), CAS RN 38363 -32-5), practolol (N-[4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]phenyl]acetamide, CAS RN 6673-35-4); triprenolol hydrochloride (hydrochloride (±) 1-[(1methylethyl)amino]-3-[2-(methylthio)phenoxy]propanol, CAS RN 39832-43-4), tolamolol (4-[2-[[2hydroxy-3- (2-methylphenoxy)propyl]amino]ethoxyl]benzamide, CAS RN 38103-61-6), bopindolol, indenolol, pindolol, propanolol, tertatolol and tilizolol, and the like; calcium channel blockers such as amlodipine besylate salt (such as 3-ethyl-5-methyl-2-(2-aminoethoxymethyl)-4-(2-chlorophenyl)-1,4-dihydro-6-methyl-3,5-pyridinedicarboxylate benzenesulfonate , e.g. Norvasc®, Pfizer), klentiazem maleate ((Z)-2-butenedioate 3-(acetyloxy)-8-chloro-5-[2-(dimethylamino)ethyl]-2,3-dihydro-2-(4methoxyphenyl )-(2S-cis)-1,5-benzothiazepin-4(5H)-one, (1:1), see also US4567195), isradipine ((±)-4(4-benzofurazanil)-1,4-dihydro- 4-(4-Benzofurazanyl)-1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylic acid methyl-1-methylethyl ester 2,6-dimethyl-3,5-pyridinedicarboxylate, see also US4466972) ; nimodipine (such as isopropyl-(2-methoxyethyl)-1,4-dihydro-2,6-dimethyl-4-(3nitrophenyl)-3,5-pyridinedicarboxylate, e.g. Nimotop®, Bayer), felodipine (such as ethylmethyl4- (2,3-dichlorophenyl)-1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylate, e.g. Plendil® extended release, AstraZeneca LP), nilvadipine (3-methyl-5-(1-methylethyl ) 2-cyano-1,4-dihydro-6-methyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylic acid ester, see also US3799934), nifedipine (such as 1,4-dihydro-2 dimethyl ester ,6-dimethyl-4-(2-nitrophenyl)-3,5pyridinedicarboxylic acid, e.g. Procardia XL® extended release tablets, Pfizer), diltiazem hydrochloride (such as (+)-cis-3-(acetyloxy)-5 monohydrochloride [2(dimethylamino)ethyl]-2,-3-dihydro-2(4-methoxyphenyl)-1,5-benzothiazepin-4(5H)-one, e.g. Tiazac®, Forest), verapamil hydrochloride (such as hydrochloride ( alpha)-[[3-[[2-(3,4dimethoxyphenyl)ethyl]methylamino]propyl]-3,4-dimethoxy-(alpha)-(1-methylethyl)benzeneacetonitrile, e.g. mer, Isoptin® SR, Knoll Labs), teludipine hydrochloride (diethyl ester monohydrochloride 2-[(dimethylamino)methyl]4-[2-[(IE)-3-(1,1-dimethylethoxy)-3-oxo-1 -propenyl]phenyl]-1,4dihydro-6-methyl-3,5-pyridinedicarboxylic acid) CAS RN 108700-03-4), belfosdil (tetrabutyl ester of [2-(2-phenoxyethyl)-1,3-propane- diyl]bis-Phosphonic acid, CAS RN 103486-79-9), fostedyl ([[4-(2-benzothiazolyl)phenyl]methyl]phosphonic acid diethyl ester, CAS RN 75889-62-2), aranidipine, azelnidipine, barnidipine, benidipine, bepridil, cinaldipine, clevidipine, efonidipine, gallopamil, lacidipine, lemyldipine, lercanidipine, monatepil maleate ((Z)-2-butenedioate (+)-N-(6,11-dihydrodibenzo(b,e)thiepin-11 -yl)4-(4-fluorophenyl)-1-piperazinebutanamide, (1:1), (±)N-(6,11-dihydrodibenzo (b,e)thiepin-11-yl)-4-(p- fluorophenyl)-1-piperazine butyramide (1:1) CAS RN 132046-06-1), nicardipine, nizoldipine, nitrendipine, manidipine, pranidipine, and the like; calcium T-channel antagonists such as mibefradil; angiotensin converting enzyme (ACE) inhibitors such as benazepril, benazepril hydrochloride (such as 3-[[1-(ethoxycarbonyl)-3phenyl-(1S)-propyl]amino]-2,3,4,5-tetrahydro-2- oxo-1H-1-(3S)-benzazepine-1-acetic acid, e.g. Lotrel®, Novartis), captopril (such as 1-[(2S)-3-mercapto-2-methylpropionyl]-L-proline, e.g. , captopril, Mylan, CAS RN 62571-86-2 and others described in US4046889), cerapril (and others described in US4452790), cetapril (alacepril, dainippon described in Eur. Therap. Res. 39:671 (1986) ; 40:543 (1986)), cilazapril (Hoffman-LaRoche) described in J. Cardiovasc. Pharmacol. 9:39 (1987), indalapril (delapril hydrochloride (3-bicyclo[2.2.1]hept-5-en-2-yl-6-chloro-3,4-dihydro-2H1,2 1,1-dioxide ,4-Benzothiadiazine-7-sulfonamide, CAS RN 2259-96-3); described in US4385051), enalapril (and others described in US4374829), enalopril, enaloprilat, fosinopril, ((such as 4cyclohexyl-1-[sodium salt [[2-methyl-1-(1-oxopropoxy)propoxy](4-phenylbutyl)phosphinyl]acetyl]-E-proline,

- 46 042294 e.g. monopril, Bristol-Myers Squibb and others described in US4168267), fosinopril sodium (4cyclohexyl-1-[[(R)-[(1 S)-2-methyl-1-(1-oxopropoxy)propoxy )-b-proline, imidapril, indolapril (Schering, described in J. Cardiovasc. Pharmacol. 5:643, 655 (1983)), lisinopril (Merck), losinopril, moexipril, moexipril hydrochloride (monohydrochloride (3S)-2-[ (2S)-2-[[(1S)-1-(ethoxycarbonyl)-3phenylpropyl]amino]-1-oxopropyl]-1,2,3,4-tetrahydro-6,7-dimethoxy-3-isoquinolinecarboxylic acid, CAS RN 82586-52-5), quinapril, quinaprilat, ramipril (Hoechsst) described in EP 79022 and Curr. Ther. Res. 40:74 (1986), perindopril erbumine (such as 2S,3aS,7aS-1-[(S)-N[(S)-1-carboxybutyl]alanyl]hexahydroindolinecarboxylic acid 1-ethyl ester, compound with tert-butylamine (1:1), e.g. Aceon®, Solvay), perindopril (Servier, described in Eur. J. clin. Pharmacol. 31:519 (1987)), quanipril (described in US4344949), spirapril (Schering, described in Acta Pharmacol Toxicol 59 (Supp. 5): 173 (1986)), tenocapril, trandolapril, zofenopril (and others described in US4316906), rentiapril (fentiapril described in Clin. Exp. Pharmacol. Physiol. 10:131 ( 1983)), pivopril, YS980, teprotide (BPP9a bradykinin enhancer CAS RN 35115-60-7), BRL 36378 (Smith Kline Beecham, see EP80822 and EP60668), MC-838 (Chugai, see CA 102:72588v and Jap J Pharmacol 40:373 (1986), CGS 14824 (Ciba-Geigy, 3-([1-ethoxycarbonyl-3-phenyl-(1S)-propyl]amino)-2,3,4,5-tetrahydro -2-oxo-1-(3S)benzazepine-1-acetic acid HCl, see UK Patent No. 2103614), CGS 16,617 (Ciba-Geigy, 3(S)-[[(1S)-5-amino-1 -carbo xypentyl]amino]-2,3,4,-5-tetrahydro-2-oxo-1H-1-benzazepine-1-ethanoic acid, see US4473575), Ru 44570 (Hoechst, see Arzneimittelforschung 34:1254 (1985) ), R 31-2201 (Hoffman-LaRoche see FEBS Lett. 165:201 (1984)), CI925 (Pharmacologist 26:243, 266 (1984)), WY-44221 (Wyeth, see J. Med. Chem. 26:394 (1983)), and described in US2003006922 (paragraph 28 ), US4337201, US4432971 (phosphonamidates); neutral endopeptidase inhibitors such as omapatrilat (Vanlev®), CGS 30440, cadoxatril and ecadotril, fazidotril (also known as aladotril or alatriopril), sampatrilate, mixanpril and hemopatrilat, AVE7688, ER4030 and described in US5362727, US5366973, US5225401, US472228 . endothelin antagonists such as tezosentan, A308165 and YM62899, and the like; vasodilators such as hydralazine (apresolin), clonidine (clonidine hydrochloride (N-(2,6dichlorophenyl)4,5-dihydro-1H-imidazole-2-amine monohydrochloride, CAS RN 4205-91-8), catapres, minoxidil ( loniten), nicotinyl alcohol (roniasol), diltiazem hydrochloride (such as (+)-cis-3(acetyloxy)-5[2-(dimethylamino)ethyl]-2,-3-dihydro-2(4-methoxyphenyl)- 1,5-benzothiazepin-4(5H)-one, eg Tiazac®, Forest), isosorbitol dinitrate (such as 1,4:3,6-dianhydro-O-glucite 2,5-dinitrate eg Isordil® Titradose® , Wyeth-Ayerst), sosorbide mononitrate (such as 1,4:3,6-dianhydro-Oglucito-1,5-nitrate, organic nitrate, such as Ismo®, Wyeth-Ayerst), nitroglycerin (such as trinitrate 2,3 -propanetriol, e.g. Nitrostat® Parke-Davis), verapamil hydrochloride (such as (±)-(alpha)[3-[[2-(3,4-dimethoxyphenyl)ethyl]methylamino]propyl]-3,4- dimethoxy-(alpha)-( 1 methylethyl)benzeneacetonitrile, e.g. Extended Release Covera HS®, Searle), chromonar (which and can be obtained as described in US3282938), clonitate (Annalen 1870 155), droprenylamine (which can be obtained as described in DE2521113), lidoflazin (which can be obtained as described in US3267104); prenylamine (which can be prepared as described in US3152173), propyl nitrate (which can be prepared as described in French Patent No. 1103113), myoflazin hydrochloride (3-(aminocarbonyl) dihydrochloride)4-[4,4-bis(4-fluorophenyl )butyl]-N-(2,6-dichlorophenyl)-1piperazineacetamide, CAS RN 83898-67-3), myxidine (benzenethanamine 3,4-dimethoxy-N-(1-methyl-2pyrrolidinylidene)-pyrrolidine, 2-[( 3,4-dimethoxyphenethyl)imino]-1-methyl-1-methyl-2-[(3,4dimethoxyphenethyl)imino]pyrrolidine, CAS RN 27737-38-8), molsidomine (internal salt 5[(ethoxycarbonyl)amino]- 3-(4-morpholinyl)-1,2,3-oxadiazolium, CAS RN 25717-80-0), isosorbitol mononitrate (1,4:3,6-dianhydro-D-glucite 5-nitrate, CAS RN 16051-77 -7), erythrityl tetranitrate ((2R, 3S)-rel-1,2,3,4-butantetrol tetranitrate, CAS RN 7297-25-8), clonitrate (3-chloro-1,2propanediol dinitrate (7Cl, 8Cl, 9Cl) CaS RN 2 612-33-1), dipyridamole 2,2',2,2'-[(4,8-di-1-piperidinylpyrimido[5,4-d]pyrimidin-2,6-diyl)dinitrilo]tetrakisethanol CAS RN 58-32-2), nicorandil (CAS RN 65141-4 6-03), pyridinecarboxamide (methyl-2-methylpropyl ester of 1,4dihydro-2,6-dimethyl-4-(2-nitrophenyl)-N-[2-(nitrooxy)ethyl]-nizoldipine-3,5-pyridinedicarboxylic acid, CAS RN 63675 -72-9), nifedipine (1,4-dihydro-2,6-dimethyl-4-(2nitrophenyl)-3,5-pyridinedicarboxylic acid dimethyl ester, CAS RN 21829-25-4), perhexyline maleate ((2Z )-2-butenedioate 2-(2,2-dicyclohexylethyl)-piperidine, (1:1) CAS RN 6724-53-4), oxprenolol hydrochloride (1-[(1-methylethyl)amino hydrochloride]-3-[2-( 2-propenyloxy)phenoxy]-2-propanol, CAS RN 6452-73-9), pentrinitrol (2,2-bis[(nitrooxy)methyl]-1,3-propanediol mononitrate (ester) CAS RN 160717-6 ), verapamil (α-[3-[[2-(3,4-dimethoxyphenyl)ethyl]-methylamino]propyl]-3,4-dimethoxy-a-(1 methylethyl)-benzolacetonitrile, CAS RN 52-53-9 ) and so on.; angiotensin II receptor antagonists such as, aprosartan, zolazartan, olmesartan, pratosartan, FI6828K, RNH6270, candesartan (2-ethoxy-1-[[2'(1H-tetrazol-5-yl)[1,1'-biphenyl]4 -yl]methyl]-1H-benzimidazole-7-carboxylic acid, CAS RN 13948159-7), candesartan cilexetil ((+/-)-1-(cyclohexylcarbonyloxy)ethyl-2-ethoxy-1-[[2'-( 1H-tetrazol-5-47 042294 yl)biphenyl-4-yl]-1H-benzimidazolecarboxylate, CAS RN 145040-37-5, US5703110 and US5196444), eprosartan (3-[1-4-carboxyphenylmethyl)-2-n- butylimidazol-5-yl]-(2-thienylmethyl)propenoic acid, US5185351 and US5650650), irbesartan (2-n-butyl-3-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl] methyl]-1,3diazaspiro[4,4]non-1-en-4-one, US5270317 and US5352788), losartan (potassium salt of 2-N-butyl-4-chloro-5hydroxymethyl-1-[(2'-( 1H-tetrazol-5-yl)biphenyl-4-yl)-methyl]imidazole, US5138069, US5153197 and US5128355), tasosartan (5,8-dihydro-2,4-dimethyl-8-[(2'-(1H- tetrazol-5-yl)[l,r-buphenyl]4-yl)methyl]pyrido[2,3-d]pyrimidin-7(6H)-one, US5149699), telmisartan (4'-[(1,4- dimethyl-2'-prop yl-(2,6'-bi-1Hbenzimidazole)-r-yl)]-[1,1'-biphenyl]-2-carboxylic acid, CAS RN 144701-48-4, US5591762), milfasartan, abitezartan, valsartan ( Diovan® (Novartis), (S)-N-valeryl-N-[[2'-(1H-tetrazol-5-yl)biphenyl-4yl)methyl]valine, US5399578), EXP-3137 (2-N-butyl -4-chloro-1-[(2'-(1H-tetrazol-5-yl)biphenyl-4yl)methyl]imidazole-5-carboxylic acid, US5138069, US5153197 and US5128355), 3-(2'-(tetrazol- 5-yl)-l,r-biphenyl-4-yl)methyl-5,7-dimethyl-2-ethyl-3H-imidazo[4,5-b]pyridine, 4'-[2-ethyl-4-methyl-6 -(5,6,7,8tetrahydroimidazo[1,2-a]pyridin-2-yl]-benzimidazol-1-yl]-methyl]-l,r-biphenyl]-2-carboxylic acid, 2-butyl-6 -(1-methoxy-1-methylethyl)-2-[2'-)-1H-tetrazol-5-yl)biphenyl-4-ylmethyl]quinazolin-4(3H)-one, 3[2'-carboxybiphenyl-4 -yl)methyl]-2-cyclopropyl-7-methyl-3H-imidazo[4,5-b]pyridine, 2-butyl-4-chloro-1[(2'-tetrazol-5-yl)biphenyl-4- yl)methyl]imidazolecarboxylic acid, potassium salt of 1(ethoxycarbonyloxy)ethyl ester of 2-butyl-4-chloro-1-[[2'-(1 H-tetrazol-5 -yl)[1,1'-biphenyl] 4 -yl]methyl]-1H-imidazole-5-carb ononic acid, dipotassium -5-carboxylate, methyl-2-[[4-butyl-2-methyl-6-oxo-5-[[2'-(1H-tetrazol-5-yl)-[1,1'-biphenyl]-4 -yl]methyl]-1-(6H)pyrimidinyl]methyl]-3-thiophenecarboxylate, 5-[(3,5-dibutyl-1H-1,2,4-triazol-1-yl)methyl]-2-[ 2-(1Htetrazol-5-ylphenyl)]pyridine, D,L-lysine salt of 6-butyl-2-(2-phenylethyl)-5-[[2'-(1H-tetrazol-5-yl)[1,1 'biphenyl]-4-methyl]pyrimidin-4-(3H)-one, 5-methyl-7-n-propyl-8-[[2'-(1H-tetrazol-5-yl)biphenyl-4yl]methyl] -[1,2,4]-triazolo[1,5-c]pyrimidin-2(3H)-one, 2,7-diethyl-5-[[2'-(5tetrazolyl)biphenyl-4-yl] potassium salt methyl]-5H-pyrazolo[1,5-b][1,2,4]triazole, potassium salt of ethyl ester 2-[2-butyl-4,5-dihydro-4-oxo-3-[2'- (1H-tetrazol-5-yl)-4-biphenylmethyl]-3H-imidazol[4,5c]pyridin-5-ylmethyl]benzoic acid, 3-methoxy-2,6-dimethyl-4-[[2'(1H -tetrazol-5-yl)-1,1'-biphenyl4-yl]methoxy]pyridine, 2-ethoxy-1-[[2'-(5-oxo-2,5-dihydro-1,2,4-ochadiazole -3-yl)biphenyl-4-yl]methyl]1H- benzimidazole-7-carboxylic acid, 1-[N-(2'-(1H-tetrazol-5-yl)biphenyl-4-yl-methyl)-Nvalerolylaminomethyl)cyclopentane-1-carboxylic acid, 7-methyl-2n-propyl -3-[[2'-1H-tetrazol-5yl)biphenyl-4-yl]methyl]-3H-imidazo[4.5-6]pyridine, 2-[5-[(2-ethyl-5.7- dimethyl-3H-imidazo[4,5b]pyridin-3-yl)methyl]-2-quinolinyl]sodium benzoate, 2-butyl-6-chloro-4-hydroxymethyl-5-methyl-3-[[2'(1H -tetrazol-5-yl)biphenyl-4-yl]methyl]pyridine, (tetrazol-5-yl)biphenyl-4-yl]methyl]pyrimidin-6-one-2[[[2-butyl-1-[( 4-carboxyphenyl)methyl]-1H-imidazol-5-yl]methyl]amino]benzoic acid, 4 (S)-[4(carboxymethyl)phenoxy]-N-[2(R)-[4-(2-sulphobenzamido )imidazol-1-yl]octanoyl]-L-proline, 1-(2,6dimethylphenyl)-4-butyl-1,3-dihydro-3-[[6-[2-(1H-tetrazol-5-yl) phenyl]-3-pyridinyl]methyl]-2nimidazol-2-one, 5,8-ethano-5,8-dimethyl-2-n-propyl-5,6,7,8-tetrahydro-1-[[2' (1H-tetrazol-5-yl)biphenyl-4yl]methyl]-1H,4H-1,3,4a,8a-tetrazacyclopentanaphthalen-9-one, 4-[1-[2'-(1,2,3 ,4-tetrazol-5-yl) biphen-4yl)methylamino]-5,6,7,8-tetrahydro-2-triphylquinazoline, 2-(2-chlorobenzoic k) imino-5-ethyl-3-[2'-(1Нtetrazol-5-yl)biphenyl-4-yl)methyl-1,3,4-thiadiazolin, 2-[5-ethyl-3-[2] dipotassium salt -(1H-tetrazol-5yl)biphenyl-4-yl]methyl-1,3,4-thiazolin-2-ylidene]aminocarbonyl-1-cyclopentenecarboxylic acid and 1ethoxycarbonyloxyethyl ester 2-butyl-4-[N-methyl-N -(3-methylcrotonoyl)amino]-1-[[2'(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-1H-imidazole-5-carboxylic acid described in patent publications EP475206, EP497150, EP539086, EP539713, EP535463, EP535465, EP542059, EP497121, EP535420, EP407342, EP415886, EP424317, EP435827, EP433983, EP475898, EP490820,26 EP490820

EP324377, EP323841, EP420237, EP500297, EP426021, EP480204, EP429257, EP430709, EP434249,

EP446062, EP505954, EP524217, EP514197, EP514198, EP514193, EP514192, EP450566, EP468372,

EP485929, EP503162, EP533058, EP467207 EP399731, EP399732, EP412848, EP453210, EP456442,

EP470794, EP470795, EP495626, EP495627, EP499414, EP499416, EP499415, EP511791, EP516392,

EP520723, EP520724, EP539066, EP438869, EP505893, EP530702, EP400835, EP400974, EP401030,

EP407102, EP411766, EP409332, EP412594, EP419048, EP480659, EP481614, EP490587, EP467715,

EP479479, EP502725, EP503838, EP505098, EP505111 EP513,979 EP507594, EP510812, EP511767,

EP512675, EP512676, EP512870, EP517357, EP537937, EP534706, EP527534, EP540356, EP461040,

EP540039, EP465368, EP498723, EP498722, EP498721, EP515265, EP503785, EP501892, EP519831,

EP532410, EP498361, EP432737, EP504888, EP508393, EP508445, EP403159, EP403158, EP425211,

EP427463, EP437103, EP481448, EP488532, EP501269, EP500409, EP540400, EP005528, EP028834,

EP028833, EP411507, EP425921, EP430300, EP434038, EP442473, EP443568, EP445811, EP459136,

EP483683, EP518033, EP520423, EP531876, EP531874, EP392317, EP468470, EP470543, EP502314,

EP529253, EP543263, EP540209, EP449699, EP465323, EP521768, EP415594, WO92/14468, WO93/08171,

WO93/08169,

WO93/04045,

WO93/03018,

WO91/00277,

WO93/04046,

WO91/07404,

WO91/00281,

WO91/15206,

WO92/02508,

WO91/14367,

WO92/14714,

WO92/13853,

WO92/00067,

WO92/09600,

WO91/19697,

WO92/00977,

WO92/16552,

WO91/11909,

WO92/20342,

WO93/05025,

WO91/12001,

- 48 042294

WO91/11999, WO91/15209, WO91/15479, WO92/20687, WO92/20662, WO92/20661, WO93/01177,

WO91/14679, WO91/13063, WO92/13564, WO91/17148, WO91/18888, WO91/19715, WO92/02257,

WO92/04335, WO92/05161, WO92/07852, WO92/15577, WO93/03033, WO91/16313, WO92/00068,

WO92/02510, WO92/09278, WO9210179, WO92/10180, WO92/10186, WO92/10181, WO92/10097,

WO92/10183, WO92/10182, WO92/10187, WO92/10184, WO92/10188, WO92/10180, WO92/10185,

WO92/20651, WO93/03722, WO93/06828, WO93/03040, WO92/19211, WO92/22533, WO92/06081,

WO92/05784, WO93/00341, WO92/04343, WO92/04059, US5104877, US5187168, US5149699, US5185340, US4880804, US5138069, US4916129, US5153197, US5173494, US5137906, US5155126, US5140037,

US5137902, US5157026, US5053329, US5132216, US5057522, US5066586, US5089626, US5049565,

US5087702, US5124335, US5102880, US5128327, US5151435, US5202322, US5187159, US5198438,

US5182288, US5036048, US5140036, US5087634, US5196537, US5153347, US5191086, US5190942,

US5177097, US5212177, US5208234, US5208235, US5212195, US5130439, US5045540, US5041152 and

US5210204, and their pharmaceutically acceptable salts and esters; α/β-adrenergic blockers such as nipradiol, arotinolol, amosulalol, bretylium tosylate (CAS RN: 61-75-6), dihydroergotamine mesylate (such as monomethanesulfonate (5'(a2))-9,10-dihydro-12' -hydroxy-2'-methyl-5'-(phenylmethyl)ergotaman3',6',18-trione, e.g. DHE 45® Injection, Novartis), carvedilol (such as (±)-1-(carbazol-4-yloxy )3-[[2-(o-methoxyphenoxy)ethyl]amino]-2-propanol, e.g. Coreg®, SmithKline Beecham), labetalol (such as 5-[1-hydroxy-2-[(1-methyl- 3-phenylpropyl)amino]ethyl]salicylamide, e.g. Normodyne®, Schering), bretylium tosylate (2-bromo-N-ethyl-N,N-dimethylbenzenemethanaminium salt of 4-methylbenzenesulfonic acid (1:1), CAS RN 61-75 -6), phentolamine mesylate (3-[[(4,5-dihydro-1H-imidazol-2-yl)methyl](4-methylphenyl)amino]phenol monomethanesulfonate (salt), CAS RN 6528-1), solipertine tartrate ((2R,3R)-2,3-dihydroxybutanedioate 7-[2-[4-(2-methoxyphenyl)-1piperazinyl]ethyl]-5H-1,3-dioxolo[4,5-^indole, (1:1 ) CAS RN 5591-43-5), Zolertin hydrochloride (mono 1-phenyl-4-[2-(1H-tetrazol-5-yl)ethyl]piperazine hydrochloride, (8Cl, 9Cl) CAS RN 7241-94-3) and the like; α-adrenergic receptor blockers such as alfuzocin (CAS RN: 81403-68-1), terazosin, urapidil, prazosin (Minipress®), tamsulosin, bunazocin, trimazocin, doxazosin, naftopidil, indoramin, WHP 164, XENOlO, fenspiride hydrochloride ( which can be obtained as described in US3399192), proroxan (CAS RN 33743-96-3) and labetalol hydrochloride, and combinations thereof; a2 agonists such as methyldopa, methyldopa HCL, lofexidine, thiamenidine, moxonidine, rilmenidine, quanobenz, and the like; aldosterone inhibitors and the like; renin inhibitors, including aliskiren (SPPlOO; Novartis/Speedel); angiopoietin-2 binding agents such as those described in WO03/030833; antianginal agents such as ranolazine hydrochloride (N-(2,6-dimethylphenyl)-4-[2-hydroxy-3-(2methoxyphenoxy)propyl]-1-piperazineacetamide dihydrochloride, CAS RN 95635-56-6), betaxolol hydrochloride (1-[4-[2-(cyclopropylmethoxy)ethyl]phenoxy]-3-[(1-methylethyl)amino]-2-propanol hydrochloride, CAS RN 63659-19-8), butoprosine hydrochloride ([4-[ 3-(dibutylamino)propoxy]phenyl](2-ethyl3-indolizinyl)-methanone, CAS RN 62134-34-3), cinepazet maleate ((2Z)-2-butenedioate of ethyl ester 4-[1-oxo-3- (3,4,5-trimethoxyphenyl)-2-propenyl]-1-piperazineacetic acid (1:1), CAS RN 50679-07-7), tosifen (4-methyl-N-[[[(1S)-1 -methyl-2-phenylethyl]amino]carbonyl]benzenesulfonamide, CAS RN 32295-184), verapamil hydrochloride (α-[3-[[2-(3,4dimethoxyphenyl)ethyl]methylamino]propyl]-3,4-dimethoxy monohydrochloride -a-(1-methylethyl)benzeneacetonitrile, CAS RN 152-114), molsidomine (5-[(ethoxycarbonyl)amino]-3-(4-morpholinyl)-1,2,3oxadiazolium internal salt CAS RN 25717-80-0 ) and ranolazine hydrochloride (N-(2,6-dimethylphenyl)4[2-hydroxy-3-(2-methoxyphenoxy)propyl]-1-piperazineacetamide dihydrochloride, CAS RN 95635-56-6); tosifen (4methyl-N-[[[(1S)-1-methyl-2-phenylethyl]amino]carbonyl]benzenesulfonamide, CAS RN 32295-184); adrenergic stimulants such as guanfacine hydrochloride (such as N-amidino-2-(2,6dichlorophenyl)acetamide hydrochloride, eg Tepex® tablets available from Robins); methyldopa hydrochlorothiazide (such as levo-3-(3,4-dihydroxyphenyl)-2-methylalanine) in combination with hydrochlorothiazide (such as 6-chloro-3,4-dihydro-2H-1,2 1,1-dioxide, 4-benzothiadiazine-7-sulfonamide, e.g., in combination with Aldoril® tablets available from Merck), methyldopa-chlorothiazide (such as 6chloro-2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide and methyldopa as described above, e.g. Aldoclor®, Merck), clonidine hydrochloride (such as 2-(2,6-dichlorophenylamino)-2-imidazoline hydrochloride and chlorthalidone (such as 2-chloro-5-(1-hydroxy- 3-oxo-1-isoindolinyl)benzenesulfonamide), e.g. Combipres®, Boehringer Ingelheim), clonidine hydrochloride (such as 2-(2,6dichlorophenylamino)-2-imidazoline hydrochloride, e.g. Catapres®, Boehringer Ingelheim), clonidine (1nimidazole -2-amine, N-(2,6-dichlorophenyl)4,5-dihydro-CAS RN 4205-90-7), gizaar (Merck; combination of losartan and hydrochlorothiazide), co-diovan (Novartis; combination of valsartan and hydrochlorothiazide, lotrel (Novartis; benase combination pril and amlodipine) and kaduet (Pfizer; combination of amlodipine and atorvastatin), and the agents described in US20030069221.

1.2.2.11. Remedies for the treatment of respiratory disorders.

The GCC agonist peptides described herein may be used in combination therapy with one or more of the following agents, which may be used to treat

- 49 042294 for respiratory and other disorders, including but not limited to: (1) βagonists, including but not limited to: albuterol (PRO VENTIL®, S ALBUT AMOl®, VENTOLIN®), bambuterol, biterol, clenbuterol, fenoterol , formoterol, isoetharine (BRONKOSOL®, BRONKOMETER®), metaproterenol (ALUPENT®, METAPREL®), pyrebuterol (MAXAIR®), reproterol, rimiterol, salmeterol, terbutaline (BRETHAIRE®, BRETHINE®, BRICANYL®), epinephrine, isoproterenol (ISUPREL ®), acid salt of adrenaline and tartaric acid (PRIMATENE®), ephedrine, ortsifenlin, fenoterol and isoetharine; (2) steroids, including but not limited to beclomethasone, beclomethasone dipropionate, betamethasone, budesonide, bunedosid, butixocort, dexamethasone, flunisolide, fluocortin, flucasone, hydrocortisone, methylprednisone, mometasone, prednisolone, prednisone, tipredane, thixocortal, triamcinolone, and triamcinolone acetonide ; (3) combinations of β2-agonist corticosteroids [eg, salmeterol-flucazone (AD V AIR®), formoterol budesonide (S YMBICORT®)]; (4) D4 leukotriene receptor antagonists/leukotriene antagonists/LTD4 antagonists (i.e., any compound capable of blocking, inhibiting, reducing, or otherwise disrupting the interaction of leukotrienes with the Cys LTI receptor), including but not limited to: zafhyukast, montelukast, montelukast sodium (SINGULAIR®), pranlukast, iralukast, pobilukast, SKB-106,203 and compounds described as having LTD4 antagonistic activity described in US Patent No. 5565473; (5) 5-lipoxygenase inhibitors and/or inhibitors of leukotriene biosynthesis [eg, zileuton and BAY1005 (CA registry 12825331-6)]; (6) histamine H1 receptor antagonists/antihistamines (i.e., any compound capable of blocking, inhibiting, reducing, or otherwise disrupting the interaction of histamine with its receptor), including but not limited to: astemizole, acrivastine, antazoline, azatadine, азеластин, астамизол, бромфенирамин, малеат бромфенирамина, карбиноксамин, каребастин, цетиразин, хлорфенирамин, малеат хлорфенирамина, циметидин, клемастин, циклизин, ципрогептадин, дезкарбоэтоксилоратадин, дексхлорфенирамин, диметинден, дифенгидрамин, дифенилпиралин, сукцинат доксиламина, доксиларнин, эбастин, эфлетиризин, эпинастин, фамотидин , fexofenadine, hydroxyzine, hydroxyzine, ketotifen, levocabastine, levocetirizine, levocetirizine, loratidine, meclizine, mepyramine, mekvuitazin, methdilazine, mianserin, mizolastine, noberastine, norasternisol, noraztemizole, phenindamine, pheniramine, picumast, promethazine, ranidine, teminidine, laminite , terfenadine, trimeprazine, tripelenamin and triprolid n; (7) anticholinergics, including but not limited to: atropine, benzatropine, biperiden, flutropium, chioscyamine (e.g., Levsin®; Levbid®; Levsin/SL®, Anaspaz®, Levsinex timecaps®, NuLev®), ilutropium, ipratropium, ipratropium bromide, metscopolamine, oxybutynin, rispenzepine, scopolamine, and tiotropium; (8) antitussives, including but not limited to: dextromethorphan, codeine, and hydromorphone; (9) decongestants, including but not limited to: pseudoephedrine and phenylpropanolamine; (10) an expectorant, including but not limited to: guaifenesin, guaicol sulfate, terpine, ammonium chloride, glycerol guaicolate, and iodinated glycerol; (11) bronchodilators, including but not limited to: theophylline and aminophylline; (12) anti-inflammatory agents, including but not limited to: fluribiprofen, diclofenac, indomethacin, ketoprofen, S-ketoprofen, tenoxicam; (13) PDE (phosphodiesterase) inhibitors, including but not limited to those described herein; (14) recombinant humanized monoclonal antibody [eg, xolair (also called omalizumab), rh µmab, and talizumab]; (15) humanized lung surfactants, including recombinant forms of SP-B, SP-C, or SP-D surfactant proteins [eg, SURFAXIN® formerly known as dsc-104 (Discovery Laboratories)], (16) epithelial sodium channel inhibitors (ENaC) such as amiloride and related compounds; (17) antimicrobials used to treat lung infections such as acyclovir, amikacin, amoxicillin, doxycycline, trimethoprine sulfamethoxazole, amphotericin B, azithromycin, clarithromycin, roxithromycin, clarithromycin, cephalosporins (ceffoxitin, cefmetazole, etc.), ciprofloxacin, ethambutol, gentimicin, ganciclovir, imipenem, isoniazid, itraconazole, penicillin, ribavirin, rifampin, rifabutin, amantadine, rimantadine, streptomycin, tobramycin, and vancomycin; (18) agents that activate chloride secretion through Ca++-dependent chloride ion channels (such as purinergic receptor agonists (P2Y(2)); (19) agents that reduce sputum viscosity, such as human recombinant DNase 1, (Pulmozyme®) ; (20) nonsteroidal anti-inflammatory drugs (acemethacin, acetaminophen, acetylsalicylic acid, aclofenac, alminoprofen, apazon, aspirin, benoxaprofen, bezpiperilone, bucloxic acid, carprofen, clidanac, diclofenac, diclofenac, diflunisal, diflunisal, etodolac, fenbufen, fenbufenac, fenclofenac fenclosic acid, fenoprofen, fentiazac, feprazon, flufenamic acid, flufenisal, flufenisal, fluprofen, flubiprofen, flubiprofen, furofenac, ibufenac, ibuprofen, indomethacin, indomethacin, indoprofen, isoxepac, isoxicam, ketoprofen, ketoprofen, ketorolac, meclofenamic acid, mefenamic acid, mefenamic acid, miroprofen, mofebutazone, nabumetone oxaprozin, naproxen, naproxen, niflumic acid ta, oxaprozin, oxpinac, oxyphenbutazone, phenacetin, phenylbutazone, phenyl

- 50 042294 butazone, piroxicam, piroxicam, pirprofen, pranoprofen, sudoxicam, tenoxicam, sulfasalazine, sulindac, sulindac, supprofen, thiaprofenic acid, thiopinac, thioxaprofen, tolfenamic acid, tolmetin, tolmetin, zidomethacin and zomepirac); and (21) aerosolized antioxidant drugs such as S-nitrosoglutathione.

1.2.2.12. Antidiabetic agents.

The GCC agonist peptides described herein may be used in therapeutic combination with one or more antidiabetic agents, including but not limited to: PPARy agonists such as glitazones (e.g., WAY-120,744, AD 5075, balaglitazone, ciglitazone, darglitazone ( CP-86325, Pfizer), englitazone (CP-68722, Pfizer), isaglitazone (MIT/J&J), MCC-555 (Mitsibishi described in US5594016), pioglitazone (e.g., such as Actos™ pioglitazone; Takeda), rosiglitazone (Avandia ™; Smith Kline Beecham), rosiglitazone maleate, troglitazone (Rezulin® described in US4572912), rivoglitazone (CS-O1 1, Sankyo), GL-262570 (Glaxo Welcome), BRL49653 (described in WO98/05331), CLX-0921 , 5-BTZD, GW-0207, LG-100641, JJT-501 (JPNT/P&U), L-895645 (Merck), R-119702 (Sankyo/Pfizer) , NN-2344 (Dr. Reddy/NN), YM -440 (Yamanouchi), LY-300512, LY-519818, R483 (Roche), T131 (Tularik), etc. and compounds described in US4687777, US5002953, US5741803, US5965584, US6150383, US61503 84, US6166042, US6166043, US6172090, US6211205, US6271243, US6288095, US6303640, US6329404, US5994554, WO97/10813, WO97/27857, WO97/28115, WO97/28137, WO97/27847, WO00/76488, WO03/000685, WO03/ 027112, WO03/035602, WO03/048130, WO03/055867 and their pharmaceutically acceptable salts; biguanides such as metformin hydrochloride (N,N-dimethylimidodicarbonimide diamide hydrochloride such as Glucophage™, Bristol-Myers Squibb); metformin hydrochloride with glyburide such as Glucovance™, Bristol-Myers Squibb); buformin (N-butylimidodicarbonimide diamide); etoformin (1-butyl-2-ethylbiguanide, Schering A. G.); other metformin salt forms (including those where the salt is selected from the group of acetate, benzoate, citrate, fumarate, embonate, chlorophenoxyacetate, glycolate, palmoate, aspartate, methanesulfonate, maleate, parachlorophenoxyisobutyrate, format, lactate, succinate, sulfate, tartrate, cyclohexanecarboxylate, hexanoate, octanoate, decanoate, hexadecanoate, octodecanoate, benzenesulfonate, trimethoxybenzoate, p-toluenesulfonate, adamantane carboxylate, glycoxylate, glutamate, pyrrolidone carboxylate, naphthalenesulfonate, 1-glucose phosphate, nitrate, sulfite, dithionate and phosphate) and phenformin; protein tyrosine phosphatase-1B (PTP-Ib) inhibitors such as A-401674, KR 61639, oC-060062, OC-83839, OC-297962, MC52445, MC52453, ISIS 113715 and described in WO99/585521, WO99/58518, WO99/ 58522, WO99/61435, WO03/032916, WO03/032982, WO03/041729, WO03/055883, WO02/26707, WO02/26743, JP2002114768, and their pharmaceutically acceptable salts and esters; sulfonylcarbamides such as acetohexamide (e.g. Dymelor, Eli Lilly), carbutamide, chlorpropamide (e.g. Diabinese®, Pfizer), gliamilide (Pfizer), gliclazide (e.g. Diamcron, Servier Canada Inc), glimepiride (e.g. described in US4379785, such as Amaryl, Aventis), glipentide, glipizide (eg, Glucotrol or Glucotrol XL extended release, Pfizer), glichidone, glisolamide, glyburide/glibenclamide (eg, Micronase or Prestab, Pharmacia & Upjohn, and Diabeta, Aventis), tolazamide (eg, tolinase) and tolbutamide (eg, orinase), and their pharmaceutically acceptable salts and esters; meglitinides such as repaglinide (eg Pranidin®, Novo Nordisk), KAD1229 (PF/Kissei), and nateglinide (eg Starlix®, Novartis), and their pharmaceutically acceptable salts and esters; α-glucoside hydrolase inhibitors (or glucoside inhibitors) such as acarbose (such as Precose™, Bayer, described in US4904769), miglitol (such as GLYSET™, Pharmacia & Upjohn, described in US4639436), camiglibose (methyl-6-deoxy- 6[(2R,3R,4R,5S)-3,4,5-trihydroxy-2-(hydroxymethyl)piperidino]-alpha-O-glucopyranoside, Marion Merrell Dow), voglibose (Takeda), adiposin, emiglitate, pradimycin- Q, салбостатин, CKD-711, MDL25637, MDL-73945 и MOR 14, и соединения, описанные в US4062950, US4174439, US4254256, US4701559, US4639436, US5192772, US4634765, US5157116, US5504078, US5091418, US5217877, US51091 и WO01/47528 ( polyamines); α-amylase inhibitors such as tendamistat, trestatin and A13688 and the compounds described in US4451455, US4623714 and US4273765; SGLT2 inhibitors, including those described in US6414126 and US6515117; aP2 inhibitors such as those described in US6548529; insulin secretagogues such as linogliride, A-4166, forskilin, dibutyryl cAMP, isobutylmethylxanthine (IBMX) and their pharmaceutically acceptable salts and esters; fatty acid oxidation inhibitors such as clomoxir and etomoxir, and their pharmaceutically acceptable salts and esters; A2 antagonists such as midaglysol, isaglidol, deriglidol, idazoxan, eroxan and fluparoxan, and their pharmaceutically acceptable salts and esters; insulin and related compounds (e.g., insulin mimetics) such as biota, LP-100, novarapid, insulin detemir, insulin lispro, insulin glargine, insulin zinc suspension (lente and ultralente), insulin Lys-Pro, GLP-I (1-36 ) amide, GLP-I (73-7) (insulintropin described in US5614492), LY-315902 (Lilly), GLP-I (7-36)-NH2), AL-401 (Autoimmune), specific formulations as described in US4579730, US4849405, US4963526, US5642868, US5763396, US5824638, US5843866, US6153632, US6191105 and WO85/05029, and primate, rodent or rabbit insulin, including their biologically active variants, including allelic variants, more preferably human insulin form (sources of human insulin include pharmaceutical

- 51 042294 ceutically acceptable and sterile formulations such as those available from Eli Lilly (Indianapolis, Ind. 46285) as H MKMulin™ (human insulin rDNA source), see also THE PHYSICIAN'S DESK REFERENCE, 55.sup.th Ed. (2001) Medical Economics, Thomson Healthcare (describing other useful human insulins); non-thiazolidinedione agents such as JT-501 and farglitazar (GW-2570/GI262579) and their pharmaceutically acceptable salts and esters; dual PPARa/γ agonists such as ARHO39242 (Aztrazeneca) , GW-409544 (Glaxo-Wellcome), BVT-142, CLX-0940, GW-1536, GW-1929, GW2433, KRP-297 (Kyorin Merck; 5-[ (2,4-dioxotuαzoludinyl)methyl]methoxu-N-[[4(trifluoromethyl)phenyl]methyl]benzamide), L-796449, LR-90, MK-0767 (Merck/Kyorin/Banyu), SB 219994, muraglitazar ( BMS), tezaglitazar (Astrazeneca), reglitazar (JTT-501) and described in WO99/16758, WO99/19313, WO99/20614, WO99/38850, WO00/23415, WO00/23417, WO00/23445, WO00/50414, WO01 /00579, WO01/79150, WO02/062799, WO03/004458, WO03/016265, WO03/018010, WO03/033481, WO03/033450, WO03/033453, WO03/043985, WO03 US Serial Application No.1053976 09/664598 filed September 18, 2000 by Murakami et al. Diabetes 47, 1841-1847 (1998), and their pharmaceutically acceptable salts and esters; other insulin-sensitizing drugs; VPAC2 receptor agonists; GLK modulators such as those described in WO03/015774; retinoid modulators such as those described in WO03/000249; GSK 3e/GSK 3 inhibitors such as 4-[2-(2-bromophenyl)-4-(4fluorophenyl-1H-imidazol-5-yl]pyridine and compounds described in WO03/024447, WO03/037869, WO03/037877 , WO03/037891, WO03/068773, EP1295884, EP1295885, etc., glycogen phosphorylase (HGLPa) inhibitors such as CP-368,296, CP-316,819, BAYR3401, and the compounds described in WO01/94300, WO02/20530, WO03/037864 and pharmaceutically acceptable salts or esters thereof; ATP uptake promoters such as those described in WO03/007990; TRB3 inhibitors; vanilloid receptor ligands such as those described in WO03/049702; hypoglycemic agents such as those described in WO03/015781 and WO03/040114; inhibitors of glycogen kinase kinase-3 such as those described in WO03/035663 such as those described in wO99/51225, US20030134890, WO01/24786 and WO03/059870; insulin-responsive DNA binding protein-1 (IRDBP- I) as described in WO03/057827 and the like, adenosine A2 antagonists such as those described in WO03/035639, WO03/035640 and the like, PPARδ agonists such as GW 501516, GW 590735 and the compounds described in JP10237049 and WO02/14291; dipeptidyl peptidase IV (DP-IV) inhibitors such as isoleucine thiazolidide, NVP-DPP72 8A (1-[[[2-[(5-cyanopyridin-2yl)amino]ethyl]amino]acetyl]-2-cyano-(S) -pyrrolidine described in Hughes et al, Biochemistry, 38(36), 11597-11603, 1999), P32/98, NVP-LAF-237, P3298, TSL225 (tryptophyl-1,2,3,4-tetrahydroisoquinoline-3carboxylic acid described in Yamada et al, Bioorg. & Med. Chem. Lett. 8 (1998) 1537-1540), valine pyrrolidide, TMC-2A/2B/2C, CD-26 inhibitors, FE999011, P9310/K364, VIP 0177, DPP4, SDZ 274-444, 2-cyanopyrrolidides and 4-cyanopyrrolidides as described in Ashworth et al, Bioorg. &Med. Chem. Lett., Vol. 6, no. 22, pp 1163-1166 and 2745-2748 (1996), and the compounds described in US6395767, US6573287, US6395767 (compounds disclosed include BMS-477118, BMS-471211 and BMS 538,305), WO99/38501, WO99/46272, WO99/67279, WO99/67278, WO99/61431WO03/004498, WO03/004496, ЕР1258476, WO02/083128, WO02/062764, WO03/000250, WO03/002530, WO03/002531, WO03/002553, WO03/002593, WO03/ 000180, and WO03/000181; GLP-I agonists such as exendin-3 and exendin-4 (including the synthetic exendin-4 39 aa polypeptide referred to as Exenatide®) and the compounds described in US2003087821 and NZ 504256 and their pharmaceutically acceptable salts and esters; peptides including amlintide and Symlin® (pramlintide acetate); and glycokinase activators such as those described in US2002103199 (fused heteroaromatic compounds) and WO02/48106 (isoindolin-1-one-substituted propionamide compounds).

The entire contents of all patents, patent applications and publications referred to herein are hereby incorporated by reference. However, when the contents of a patent application or publication containing precise definitions are incorporated by reference, those precise definitions are to be understood as applying to the cited patent, patent application or publication in which they are found and not to the rest of that application. In the event of a conflict, the present description, including definitions, takes precedence. The references cited herein should not be taken as prior art for the invention.

It should be understood that while the invention has been described in combination with preferred specific embodiments, the foregoing description is intended to illustrate and not limit the scope of the invention. Those skilled in the art will recognize that various changes can be made and equivalents can be replaced without departing from the scope of the invention, and furthermore that the advantages and modifications are obvious to those skilled in the art to which the invention pertains.

Examples

Example 1 Preparation of side chain protected fragments of SP-304 (as in WO2012/118972).

Coupling Fmoc-AA-OH to 2-ClTrt Resin 2-ClTrt resin (10 g, displacement=1.0 mmol/g resin) was suspended in 100 ml dichloromethane (DCM) for 5 minutes and then dried. esterification

- 52 042294 was carried out using 1.5 equivalents of Fmoc-amino acid and 1.7 equivalents of diisopropylethylamine (DIEA) in 80 ml of DCM (with a minimum amount of dimethylformamide (DMF) to completely dissolve the amino acid) for 2 hours. The resulting resin was washed with 60 ml DCM and end-capped with a solution of 60 ml DIEA/methanol (1:9, v/v) for 30 minutes. The loaded resin was then washed with DCM (6 vols) 2 times, DMF (6 vols) 3 times, and methyl tert-butyl ether (MTBE) (6 vols) 3 times, and dried under high vacuum. The replacement of the Fmoc protected resin was determined by Fmoc release assay. Finally, Fmoc deprotection was performed with a mixture of 5% piperidine, 1% 1,8 diazobicyclo[5.4.0]undec-7-ene (DBU) and 1% N-hydroxybenzotriazole (HOBt) in DMF (10 vols) 2 times, and the resin was washed and dried under high vacuum to obtain the final resin for synthesis of peptides. The results of the experiments are listed in table. VIII below.

Table VIII. Preparation of H-Gly-2ClTrt and H-Leu-2ClTrt resin

2-ClTrt resin with amino acid Synthesis scale (mmol) Resin Load Substitution by Fmoc Release Analysis Exit (mass, % output) Resin H-Gly2ClTrt 4.16 0.57 mmol/g resin 6 g, 82% Resin H-Leu- 2ClTrt 5.20 0.81 mmol/g resin 6.4 g, 100% Resin H-Gly2ClTrt 300 0.80 mmol/g resin 328.6 g, 88% Resin H-Leu- 2ClTrt 300 0.75 mmol/g resin 338.5 g, 84%

Synthesis of fragments A and B with protected side chains.

Resin H-Gly-2-ClTrt or resin H-Leu-2-ClTrt was suspended in DMF (10 volumes) for 20 minutes, then dried. The resulting resin was washed with DMF (10 volumes) for 5 minutes. Chain assembly was performed using conventional Fmoc chemistry. In general, 1.5 equivalents of Fmoc-amino acid and 1.5 equivalents of HOBt were dissolved in DMF (4.5 volumes), followed by the addition of 1.5 equivalents of DIEA. The resulting solution was then cooled to below 5°C using an ice-water bath and activated by adding 1.5 equivalents of HBTU. DCM (1.5 volumes) was added to the resin, followed by the addition of the activated Fmoc-amino acid solution. The resulting mixture was stirred at room temperature for 2 hours and the completion of the acylation was monitored by the Kaiser test. If the Kaiser test indicated the presence of an unreacted amine after 2 hours, it was necessary to reattach in the same way using 1.0 equivalents of Fmoc-amino acid, 1.0 equivalents of HOBt and 1.0 equivalents of DIEA. The capping was mainly carried out by acetylation of the unreacted amine with an acetic anhydride/pyridine/DMF solution. The peptide sequence was assembled by repeating the above capping procedure with the appropriate Fmoc amino acid derivatives in sequence from C- to N-terminus. Attachment of the Fmoc-Cys(Trt)-OH or Fmoc-Cys(Acm)-OH residue was carried out using in situ activation of 2.0 equivalents Fmoc-Cys(Trt)OH or Fmoc-Cys(Acm)-OH, 2.0 equivalents HOBt and 2.0 DIC equivalents in the DCM/DMF method to minimize cysteine racemization.

After completion of the synthesis step, the resin with the peptide was thoroughly washed with DMF (10 volumes), MTBE (10 volumes), DMF (10 volumes, 3 times) and MTBE (10 volumes, 3 times) and then dried in a vacuum oven to constant weight. .

The side chain protected peptide was cleaved from the resin using 1% TFA/DCM (10 vols) 3 times, 5 minutes each time, and the cleaved fractions were collected in pyridine each time (1:1 volume ratio to TFA in each cleaved fraction). The peptide resin was washed with DCM (7.5 volumes). The fractions were combined and concentrated in vacuo to 10% of the original volume, and the resulting solution was diluted with ethanol (3 volumes) and concentrated to 50% of the original volume. Finally, the peptide was precipitated by adding water (1 volume). The solid was collected by vacuum filtration or centrifugation and washed with water twice. The product was dried under vacuum to constant weight and analyzed by HPLC and ES-MS. The results of the experiments are presented in table. ix below.

- 53 042294

Table IX Preparation of fragments A and B

Fragment Synthesis scale (mmol) Final Peptide Resin Weight and Resin Weight Gain Yield Fragment yield obtained (% yield) Purity (HPLC) Fragment Masses Calculated/ Detected* FmocAA7-140H (1) 3.4 9.5 g, bb, 4% 3.337 g (61.3%) 87.8% 1599.95/1598.86 VosAA1-60N (2) 5, 2 11.8 g, 76.3% 5.896 g (76.9%) 94.6% 1474.80/1473.94 FmocAA7-140H (1') 200 479.7 g, 74.4% 213.7 g (66.8%) 90.1% 1599.95/1598.81 BocAAl-bOH (2') 200 544.0 g, 101.3% 247.0 g (83.7%) 94.2% 1474.80/1473.94

*calculated=average molecular weight; detected = monoisotopic mass by ES-MS.

Example 2 Condensation of SP-304 fragments in solution (as in WO2012/118972).

Fragment C synthesis: H-AA15-16OtBu (1-1) Fmoc-Cys(Acm)-OH solution (124.38 g, 0.3 mol), H-LeuOtBu.HCl (67.12 g, 0.3 mol ) and HOBt (40.54 g, 0.3 mol) in DMF (600 ml) were cooled to -5°C. 2-[1H-benzotriazol-1-yl]-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) (113.79 g, 0.3 mol) was added and completely dissolved. DIEA (183.1 ml, 1.05 mol) was added dropwise over a period of 105 minutes at the same temperature with good stirring maintaining the pH of the mixture between 6 and 7. Stirring was continued for 15 minutes at 0°C and the reaction was monitored with TLC. The reaction mixture was diluted with ethyl acetate (EtOAc) (600 ml) and 5% H ₃ PO ₄ (300 ml). The organic layer was separated and the aqueous layer was extracted with EtOAc (600 ml). The combined extracts were washed with 5% H ₃ PO ₄ (2 times), H ₂ O (1 time), saturated NaHCO ₃ (3 times), H ₂ O (2 times) and brine (2 times). The solution was dried over anhydrous MgSO ₄ , filtered and evaporated to dryness in vacuo. The product was recrystallized from petroleum ether/EtOAc (3:1) and dried: 166.75 g (95.0% yield, 99.0% purity).

Fmoc-Cys(Acm)-Leu-OtBu (166.75 g, 0.277 mol) was dissolved in a 10% piperidine/DCM solution (810 ml) with stirring. The reaction was monitored by TLC. After completion of the reaction in 3 hours, the solvent and volatiles were removed using a rotary vacuum evaporator. The resulting oily material was triturated with petroleum ether to remove by-products by decantation. The precipitate, a syrup, was taken up in EtOAc and washed with a mixture of NaH2PO4/Na2HPO4 solutions (pH 6), then saturated NaHCO ₃ , purified H ₂ O and brine. The organic layer was dried over anhydrous MgSO ₄ . Evaporation of the solvent and volatiles gave the oily product H-Cys(Acm)Leu-OtBu (1-1) (73.23 g, yield: 73.1%, purity: 98.0%).

Synthesis of fragment B-C: HAA7-16OtBu (2-3).

Solution of Fmoc-AA7-14OH (1') (198.3 g, 124.0 mmol), H-AA15-16OtBu (1-1) (52.3 g, 148.8 mmol) and Cl-HOBt (21, 0 g, 124.0 mmol) in DMF (2500 ml) was cooled to -5°C. HBTU (51.7 g, 136.4 mmol) was added and completely dissolved. DIEA (54.1 ml, 310 mmol) was then added with stirring, maintaining the pH of the mixture between 6 and 7. Stirring was continued for 30 minutes at 0-5°C. The reaction mixture was allowed to slowly warm up to 20-27°C, and stirring was continued for one and a half hours. The mixture was then poured into pre-chilled (10-20° C.) 0.5 N aqueous HCl (20 L). The suspension was kept at 20-25°C for 45 minutes. The solid was collected by filtering the suspension through a medium pore fritted glass funnel and then washing with 0.5 N aqueous HCl 3500 ml, 2 times), purified water (3500 ml), saturated aqueous NaHCO ₃ (3500 ml, 2 times) and purified water (3500 ml, 2 times), and diethyl ether (2000 ml, 2 times). Finally, the wet, crude FmocAA7-16OtBu peptide was dried in a desiccator under high vacuum at room temperature to give 238.22 g of product (85.27% purity, 98.9% yield).

To a solution of FmocAA7-16OtBu (234.88 g, 120.9 mmol) in DMF (2350 ml) was added piperidine (123 ml, 1245.2 mmol). The reaction mixture was stirred at 25°C for two and a half hours, and then the mixture was poured into n-hexane (20.0 l). The resulting sticky precipitate was triturated with n-hexane (3500 ml, 7 times). The sticky precipitate was dissolved in a minimum amount of DMF (2000 ml) and then poured into 0.5 N aqueous HCl (10 volumes). The solid was collected by filtration and washed with purified water (3 times) and then with diethyl ether (3000 ml, 3 times), air dried overnight and then vacuum dried to give the product (2-3) (HAA7-16OtBu ) (183.67 g, purity: 68.4%, yield %: 88.33%, ES-MS, MW: calculated=1721.2, found=1719.84).

Synthesis of fully protected A-B-C: BocAA1-160tBu (3-3).

A solution of HAA7-16OtBu (2-3) (183.67 g, 106.71 mmol), BocAA1-6-OH (2') (157.65 g, 106.71 mmol) and 6-Cl-HOBt (18.141 g , 106.71 mmol) in DMF (3 L) was cooled to -3-0°C. HBTU (44.523 g, 117.38 mmol) was added and completely dissolved. DIEA (55.8 ml, 320.13 mmol) was then added with stirring, maintaining the pH of the mixture at 6. Stirring was continued for 20 minutes at -5-0°C. reaction mixture

- 54 042294 si allowed to slowly warm up to 25°C, and stirring was continued for 2.5 hours, followed by additional addition of HBTU (4,048 g, 10,671 mmol) and DIEA (2 ml). Stirring was continued for the next 1.5 hours. The resulting mixture was poured into MeOH (15 L) and the precipitate was collected and washed with MeOH/DMF (5:1, v/v) (2 times, 3 L), 0.1 N HCl (3 L, 2 times) , saturated NaHCO ₃ (2 times), purified water (3 times), diethyl ether (2 times) and dried in vacuo to give the product BocAA1-16OtBu (3-3) (278.0 g, 82.0% yield). Note: purity was determined after deprotection).

Synthesis of partially protected linear SP-304: HAA1-16OH (4-4).

A mixture of TFA/TIS/EDT (8:1:1, 2400 ml) was cooled to (0-5° C.) under nitrogen and BocAA1-16OtBu (3-3) (201 g) was added in portions. The resulting suspension was stirred at 0-10°C for 30 minutes, then the reaction solution was allowed to warm to 20-25°C in a water bath (10 minutes) and stirring was continued for an additional 1 hour and 50 minutes at the same temperature. The reaction mixture was poured into pre-chilled (10° C.) MTBE (18 L). Some heat was generated during the addition of the peptide/TFA solution and the internal temperature rose to 25°C. Then the resulting suspension was kept in an ice-water bath (5-10°C) for 40 min. The precipitate was collected by filtration and washed with MTBE (2000 ml, 4 times) and dried in vacuo over P ₂ O ₅ to give 148.37 g of an off-white product, HAA1-16OH (4-4) (frequency: 62.23%, ES-MS, MW: calculated=1828.07, found=1826.67).

Example 3 Oxidative cyclization and purification of SP-304 with polystyrene absorbent resin (as in WO2012/118972).

HAA1-16OH (4-4) (0.58 g) was dissolved in 5 ml of acetonitrile and diluted with 575 ml of purified water. The solution was adjusted to pH 8-9 with 25% ammonia solution and 3% hydrogen peroxide (0.58 ml) was added, then the reaction mixture was maintained for one hour with monitoring of disulfide formation by HPLC. Nitrogen was then passed through the reaction mixture and the solution was acidified to pH 3-4 with acetic acid (71.8% by HPLC, 98.5% peak area recovery calculated). 1% iodine/ACN was added dropwise to the resulting mixture over a period of 10 minutes with good stirring until the yellow color of the iodine persisted. Stirring was continued for 30 minutes at 17-20°C. Iodine was inactivated by adding 0.5 M aqueous ascorbic acid until the yellow color disappeared. The pH of the mixture was then adjusted to 6-7 with 25% ammonia solution (51.0% by HPLC, estimated recovery of 50% of peak area).

Polystyrene absorbent resin (D101) was packed into a CM 3(ID)χ9(L) column and balanced well with column volumes (CV) of ethanol, 4 CV of purified water, 2 CV of 5% aqueous HCl, 4 CV of purified water, 2 CV of 2% aqueous NaOH and 4 CV of purified water at a flow rate of 3 CV/hr. The oxidized peptide solution was then applied to the column at 2 CV/hr. After loading, the column was washed with 2 CV of purified water at 2 CV/hour. The elution was carried out by applying 80% aqueous ethanol to the column at 2 CV/hr. Fractions with UV absorption at 215 nm were collected and pooled (125 ml). Then the combined fractions were evaporated in vacuum to 10% of the original volume, and the suspension was besieged with 125 ml of cold MTBE (10 volumes). The solid was collected by filtration and dried in vacuo to give crude SP-304 (0.282 g, 55.0% by HPLC).

Example 4 Oxidative cyclization and purification of SP-304 by RP-HPLC (as in WO2012/118972).

The crude peptide (4-4), obtained as described in example 2, was dissolved in 10% aqueous ACN to an approximate concentration of 1.25 g/l with constant stirring with a mechanical stirrer. The pH of the peptide solution was adjusted to 8.5-9.0 with 20% aqueous ammonia, and the resulting solution was vigorously stirred without a lid. Hydrogen peroxide (3%, 0.25 equivalents) was added and stirring continued at room temperature for 60 minutes. HPLC analysis showed complete consumption of the linear peptide. The solution was then acidified to pH 3-4 with 10% aqueous AcOH. The resulting solution was diluted to a concentration of approximately 1 g/l with purified water. Iodine (1.3% in ACN) was added with vigorous stirring over a period of 10 minutes while the yellow color of the iodine persisted. At approximately half hour intervals, samples were taken from the mixture and analyzed by RP-HPLC. The peak of the monocyclized peptide gradually decreased and a new peak (of the dicyclized peptide) appeared. Oxidation was completed when no monocyclized peptide peak remained. The excess iodine was neutralized with a small amount of ascorbic acid. The resulting solution was applied to a C18 RP-HPLC column packed with Kromasil 100A, 10 μm silica gel. After applying the dicyclized peptide solution, 3 column volumes of a solution of 90% mobile phase A (1.0% TEA, 0.5% H ₃ PO ₄ in H ₂ O, pH 7) and 10% mobile phase B (acetonitrile) were applied to wash application lines. A gradient was then run from 10 to 30% B over 80 minutes. Fractions were collected at recorded intervals when the main peak began to elute. The purity of each fraction was monitored by analytical RP-HPLC. Fractions of <95% purity (not meeting the main pool criteria) were pooled accordingly and processed further using the same buffer system and gradient elution parameters as above. All fractions >95% pure

- 55 042294 were combined and stored at 2-8°C. The purified peptide solution was diluted 1:1 with purified water and then applied to the same RP-HPLC column. Counterion exchange was performed by washing the column with 2-3 column volumes of 0.5 M aqueous ammonium acetate, followed by a gradient elution from 90% C (0.2% aqueous AcOH) and 10% mobile phase D (ACN) to 50% mobile phase C and 50% mobile phase D for 50 minutes. Fractions were collected at recorded intervals and monitored by analytical RP-HPLC. Fractions (>95%) were collected and lyophilized to give the final dry peptide, 68.0 g (96.1% pure).

Example 5 Desalting and isolation of SP-304 after purification by RP-HPLC (as in WO2012/118972).

After purification of plecanatide by RP-HPLC as described in Example 4, it was desalted and isolated. Briefly, purified plecanatide in ammonium acetate/acetonitrile/water buffer was applied to a column packed with a polymeric absorbent (macroporous adsorption resin) and then eluted with an alcohol/water mixture. Finally, the peptide alcohol solution was concentrated under reduced pressure, precipitated with an ether such as diethyl ether or MTBE, and dried in vacuo to give the final product.

Selection of resins (polymeric adsorbents).

Resin pre-treatment: Polymer adsorbents, DA201-C (from Jiangsu Suqing, China; cross-linked polystyrene; surface area 1200-1400 m ² /g; average pore diameter: 3-4 nm; pore volume: 1.1-1.2 ml/g Bulk density: 0.68-0.75 g/ml Specific gravity: 1.03-1.1 g/ml Humidity: 50-60% Particle size: 0.315-1.25 mm >95 %; effective diameter: 0.4-0.7 mm; uniformity coefficient: <1.6%), DA201-H (from Jiangsu Suqing, China; cross-linked polystyrene; surface area >800 m ² /g; average pore diameter : 6-8 nm Pore volume: 1.5-1.8 ml/g Bulk density: 0.65-0.70 g/ml Specific gravity: 1.02-1.07 g/ml Humidity: 55-65%, particle size: 0.315-1.25 mm >95%, effective diameter: 0.4-0.7 mm, uniformity coefficient: <1.6%), ADS-5 (from Nankai Hecheng, China; cross-linked polystyrene; surface area 520-600 m ² /g; average pore diameter: 25-30 nm; bulk density: 0.7-0.8 g/ml; humidity: 60-70%; particle size: 0.315-1 .25 mm >95% Uniformity coefficient: <1 .6%), and ADS-8 (from Nankai Hecheng, China; cross-linked polystyrene; surface area 450-500 m ² /g; average pore diameter: 12-16 nm; bulk density: 0.65-0.75 g/ml; humidity: 60-70%; particle size: 0.315~1.25mm >95%; homogeneity coefficient: <1.6%) suspend in 4-6 volumes of ethanol overnight. Decant or aspirate the supernatant from the settled resin. Add 68 volumes of deionized water and resuspend the resin with gentle agitation from above. Decant again or suck off the supernatant from the settled resin. Repeat the above watering and decanting steps until the appearance of fine particles is minimal.

Column Packing and Regeneration: Resuspend the above pre-treated resins with 1-2 volumes of deionized water to form the appropriate resin slurry using gentle shaking. Slowly pour the resin slurry from the bottom into the column to prevent air entrapment. After the resin slurry has been completely transferred to the column, flush the inside of the column using a spray bottle containing deionized water. Open the outlet of the column to form a settled resin layer (ID=4 cm, H=10 cm). The resin layers were then washed successively at a flow rate of 3 CV per hour with 4 CV deionized water, 2 CV 5% aqueous HCl, 4 CV deionized water, 2 CV 2% aqueous NaOH, and finally 4 CV deionized water until the pH of the eluate was approximately 7.

Preparation of application samples: 2000 mg of lyophilized plecanatide was dissolved in a mixture of 60 ml of ACN and 150 ml of 0.2% aqueous AcOH (pH of aqueous AcOH was adjusted to 4 with 10% aqueous ammonia). After filtration with a 1.2 μm nylon membrane, the filtrate was diluted to 250 ml with 0.2% aqueous AcOH (pH4) and divided into 4 portions (62.5 ml each) for application.

Application of samples to the columns: 62.5 ml of the above peptide solution was applied to 4 of the above columns at a flow rate of 2CV/h, respectively. The eluate after application was collected and tested by RP-HPLC to evaluate the absorbent capacity of each resin. The absorbent capacity of each resin is shown in Table 1. X below.

Table X

resins Peptide in the applied sample Absorbed peptide Absorption ratio DA201-C 50 0 mg 303.5 mg 60.7% DA201-H 50 0 mg 493.4 mg 98.7% ADS-5 50 0 mg 450.5 mg 90.1% ADS-8 50 0 mg 4 6 6.1 mg 93.2%

- 56 042294

HPLC method: HPLC device: Shimadzu LC-10AD vp; column: Kromasil, C18, 4.6x250 mm; mobile phase A: 0.1% TFA in water; mobile phase B: 0.1% TFA in ACN; detection at: 215 nm; column temperature: 40°C; flow rate: 1.0 ml/min; gradient: 25% B to 45% B over 30 min.

Absorbent Capacity Calculation: The absorbent capacity of each resin was shown by the absorbance ratio of the peptide applied to each column, which was calculated by the amount of peptide absorbed on each resin column divided by the amount of peptide in each applied sample (500 mg). The amount of peptide absorbed on each column was calculated using the formula below:

Amount of peptide absorbed = Amount of peptide in the applied sample - Amount of peptide in the eluate after application = 500 mg-62.5 mlx (1.6 mg/mlx HPLC peak area for eluate/HPLC peak area for peptide standard solution).

Rinse the column with deionized water.

The above loaded columns were then washed with 2CV deionized water at 2CV/hour to remove salts. Wash eluates were collected and analyzed by RP-HPLC to determine the amount of peptide desorbed from water using the same method described above.

The ratio of desorbed peptides for each resin are listed in table. XI below.

Table XI

resins Peptide adsorbed on resin Desorbed peptide Desorption ratio DA201-C 303.5 mg 184.5 mg 60.8% DA201-H 493.4 mg 4 0.9 mg 8.3% ADS-5 450.5 mg 41.9 mg 9.3% ADS-8 4 6 6.1 mg 4 0.1 mg 8.6%

Peptide desorption with 90% ethanol/water: After washes with 2CV water, the peptide absorbed on each column was then eluted with 1-2CV 90% ethanol in water at 2CV/hr. The eluate was collected and analyzed by RP-HPLC to determine the amount of peptide desorbed with 90% ethanol using the same method described above. The ratio of desorbed peptides for each resin are listed in table. XII below.

Table XII

resins Peptide adsorbed on resin after washes with 2CV water Desorbed peptide Desorption ratio DA201-C 119.0 mg 47.6 mg (via 2CV ethanol) 40% DA201-H 452.5 mg 452.5 mg (via 1.5CV ethanol) 100% ADS-5 408.6 mg 408.6 mg (via 1.5 CV ethanol) 100% ADS-8 426.0 mg 426.0 mg (via 1.5 CV ethanol) 100%

Isolation of the peptide from the ethanol solution.

The peptide/ethanol/water solution collected from each column was concentrated under reduced pressure, precipitated with MTBE, filtered and dried in vacuo to obtain the final product. The total yield of the peptide processed on each column is shown in table. XIII below.

- 57 042294

Table XIII

resins Peptide in the applied sample final product received total output DA201-C 50 0 mg 52 mg 10.4% DA201-H 50 0 mg 4 60 mg 92.0% ADS-5 50 0 mg 400 mg 80.0% ADS-8 50 0 mg 430 mg 86.0%

Conclusion from the selection of resin.

According to the above data (Table X-XIII), DA201-H resin showed the best absorbent capacity and the best desorption efficiency (with ethanol) for plecanatide among the resins in the experiment. Optimization of Desalting and Recovery Method Selection of Elution Solvents: Isopropanol and ethanol are two common solvents for peptide elution from polymeric absorbents. In table. XIV shows the amount of plecanatide that can be dissolved in an aqueous solution of ethanol or isopropanol, depending on the vol./about. % isopropanol (or ethanol): water.

Table XIV

Solvent Solubility 90%1RA/water 67.5 mg/ml 75%1RA/water 596.1 mg/ml 50%1RA/water 635.0 mg/ml 90%ETOH/water 302.0 mg/ml 75%ETOH/water 700.0 mg/ml

Water from the peptide/alcohol solution can be removed by azeotropic distillation. In table. XV shows the properties of binary azeotropic mixtures of ethanol/water and isopropanol/water.

Table XV

Component A Component B Boiling point A Boiling point B Boiling point of an azeotropic mixture wt.% And in the azeotropic mixture Water ethanol 100°C 78.3°C 78.2°C 4% Water Isopropanol 100°С 82.3°С 80.3°С 12.6%

Degradation of plecanatide may occur during long term storage of the peptide/alcohol/water solution and the concentration process. In table. XVI shows stability data for plecanatide in 75% IPA/water and 90% EtOH/water at 23°C.

Table XVI

Duration Purity (in 90% aqueous EtOH)* Purity (in 75% aqueous IPA)* 0 hours 98.5% 98.7% 2 hours 98.4% 98.7% 4 hours N/A 98.3% 6 hours 98.1% 97.5% 8 ocloc'k N/A 96.6% 10 hours 95.5% N/A 24 hours 92.0% N/A 25 hours N/A 96.1%

* HPLC method: HPLC device: Shimadzu LC-10AD vp: column: Kromasil, C18, 4.6x250 mm; mobile phase A: 0.1% TFA in water; mobile phase B: 0.1% TFA in ACN; detection at: 215 nm; column temperature: 40°C; flow rate: 1.0 ml/min; gradient: 25%B to 45%B over 30 min.

According to the obtained test data, plecanatide was quite stable at 23 °C in alcohol/water solution for 6 hours.

Elution experiments were carried out using isopropanol/water mixtures of various v/v%. The desorption ratio and water content in the peptide eluates are listed in Table 1. XVII below.

Table XVII

v/v% IPA/water 75% IPA/water 95% IPA/water 100% IPA/water Water content in peptide eluate* 65.9% 53.0% 49.8% Desorption ratio 100% 100% 90%

75% isopropanol/water as elution solution: 500 mg of plecanatide (98.1% pure) was dissolved in a mixture of 16 ml of ACN and 49 ml of 0.2% aqueous AcOH (the pH of the 0.2% AcOH solution was adjusted to 4.0 by adding 10% aqueous NH4OH). After filtration through a 1.2 μm nylon membrane, the peptide solution was applied to a column packed with DA201-H resin (ID=4 cm, H=10 cm, packed and pretreated in the manner mentioned earlier) at 2CV/h. After loading, the column was washed with 2 CV of deionized water at 2 CV/hour. The peptide was then eluted with 1.5 CV of 75% IPA/water at 2CV/hour. The elution was monitored by RP-HPLC. The eluate was collected (124 ml, 98.3% pure). Karl Fischer analysis showed water (65.9% by weight). Into a 1-necked, 500 ml round bottom flask was placed 124 ml of the peptide/IPA/water eluate collected above (97.6% pure, slight degradation occurred during storage at 2-8°C for 2 days). The flask was then placed in a rotary evaporator under reduced pressure (60 Pa) and partially immersed in a water bath at 23°C. An off-white suspension was obtained by pushing about 622 ml of isopropanol over 2 hours to about 1/3 of the original solution volume (97.7% pure). Karl Fischer analysis showed water (0.17% wt.%). 350 ml of pre-cooled diethyl ether was added to the concentrate, the solid was collected by centrifugation at 3500 rpm for 3 minutes and dried in vacuo to give 333 mg of the final product (yield 6 6.6%, purity 97.2% by HPLC).

95% isopropanol/water as elution solution I: 500 mg of plecanatide (purity 98.1%) was dissolved in a mixture of 16 ml of ACN and 49 ml of 0.2% aqueous AcOH (pH of a solution of 0.2% AcOH was adjusted to 4.0 with addition of 10% aqueous NH4OH). After filtration through a 1.2 μm nylon membrane, the peptide solution was applied to a column packed with DA201-H resin (ID=4 cm, H=10 cm, pretreated in the manner mentioned earlier) at 2CV/hr. After loading, the column was washed with 2 CV of deionized water at 2 CV/hour. The peptide was then eluted with 1.5 CV of 95% IPA/water at 2CV/hour. The elution was monitored by RP-HPLC. The eluate was collected (117 ml, 98.1% purity). Karl Fischer analysis showed water (52% wt.%). Into a 1-necked, 500 ml round bottom flask was placed 117 ml of the peptide/IPA/water eluate collected above. Then the flask was placed in a rotary evaporator under reduced pressure (50 Pa) and partially immersed in a water bath at 23°C. An off-white solid was obtained by sweeping approximately 470 ml of isopropanol over 130 minutes (purity 97.9%). To the above solid was added 50 ml of pre-cooled diethyl ether to form a suspension and evaporated under reduced pressure (50 Pa) on a rotary evaporator at 23° C. to dryness. The yield was 430 mg of the final product (86%). HPLC purity was 97.9%.

95% isopropanol/water as elution solution II: 500 mg of plecanatide (purity 98.1%) was dissolved in a mixture of 16 ml of ACN and 49 ml of 0.2% aqueous AcOH (the pH of the 0.2% AcOH solution was adjusted to 4.0 with addition of 10% aqueous NH4OH). After filtration through a 1.2 μm nylon membrane, the peptide solution was applied to a column packed with DA201-H resin (ID=4 cm, H=10 cm, packed and pre-treated in the manner mentioned earlier) at 2 CV/h. After loading, the column was washed with 2 CV of deionized water at 2 CV/hour. The peptide was then eluted with 1.5 CV of 95% IPA/water at 2 CV/hour. The elution was monitored by RP-HPLC. The eluate was collected (118 ml, 98.2% purity). Karl Fischer analysis showed water (53% wt.%). To a 1-necked, 500 ml round bottom flask was placed 118 ml of the peptide/IPA/water eluate collected above. Then the flask was placed in a rotary evaporator under reduced pressure (50 Pa) and partially immersed in a water bath at 23°C. An off-white suspension (~40 ml) was obtained by pushing about 330 ml of isopropanol over 100 minutes (97.7% purity). 400 ml of pre-cooled diethyl ether was added to the above suspension to form a suspension. After maintaining at ambient temperature for 1 hour, the solid was collected by centrifugation at 3500 rpm for 3 minutes and dried in vacuo to give 370 mg of final pro

- 59 042294 duct. The yield was 74%. HPLC purity was 97.9%.

95% isopropanol/water as elution solution III: 10 g of plecanatide was desalted and precipitated in a manner similar to that described above. Interestingly, the precipitation yield improved to 93%, which is a significant improvement in yield. HPLC purity after precipitation was 98.47%.

Example 6 Characterization of lyophilized SP-304 and precipitated SP-304 (as in WO2012/118972).

Plecanatide purified by lyophilization as described in Example 4 and plecanatide purified by precipitation as described in Example 5 were analyzed to determine the values of significant chemical impurities such as levels of acetamide, TFA, ammonium ions and acetonitrile. The results are listed in the table below.

Acetamide TFA Ammonium ions Acetonitrile Lyophilized plecanatides 356ppm 0.14% 1.58% 40ppm besieged plecanatid <28ppm (LOQ way) 0.09% 0.23% Not detected (LOQ 20ppm)

As shown by the above results, the precipitation process provides significantly reduced levels of undesirable process impurities.

Plecanatide purified by lyophilization as described in Example 4 and plecanatide purified by precipitation as described in Example 5 were measured to obtain their bulk density, bulk density, particle size distribution and shape.

Equipment.

(1) Bulk Density Tester Model TD-1020;

(2) Acoustic screen separator model L3P;

(3) Optical microscope LINITRON 2850.

Ways.

1) Bulk and bulk density measurements: modified USP method 1;

a. A 100.0 ml graduated cylinder was used for lyophilized plecanatide,

b. A 10.0 ml graduated cylinder was used for precipitated plecanatide.

2) Analysis of particle size distribution;

a. used sieves: 200, 140, 100, 60, 40 and 30 mesh,

b. sample size: 2 g lyophilized plecanatide and 6.4 g precipitated plecanatide.

3) Optical microscopic analysis: particle size and shape;

a. the dry powder was manually distributed on a microscope slide,

b. magnification: 100 x,

c. normal lighting conditions (without polarizing filters).

Results.

(1) Physical properties: lyophilized plecanatide is a light, fluffy and white powder. Precipitated plecanatide is a slightly off-white powder.

(2) Bulk and bulk density: in the table. XVIII presents bulk and bulk density data for plecanatide samples, both lyophilized and precipitated.

Table XVIII

Plecanatide sample Bulk density, g/ml Bulk density, g/ml Freeze-dried, lot 101221 0.0332 0.0680 besieged, party 120210 0.486 0.641

As can be seen from the data, the precipitated plecanatide is surprisingly significantly denser than the lyophilized plecanatide. Precipitated plecanatide has a lower tendency to generate dust during processing, which provides the benefits of increased safety and reduced processing losses.

(3) Particle size distribution: In the table. XIX summarized the particle size distribution analysis. In FIG. 1 the data is presented graphically.

- 60 042294

Table XIX

# mesh size Particle size (µm) Retained weight (g) Interest withheld Lyophilized besieged Lyophilized Besieged thirty 600 0.07 0.45 3.6% 7.1% 40 425 0.26 0.17 13.3% 2.7% 60 250 0.17 0.67 8.7% 10.5% 100 150 0.51 2.08 26.0% 32.7% 140 106 0.65 1.22 33.2% 19.2% 200 75 0.24 0.68 12.2% 10.7% Pallet <75 0.06 1.09 3.1% 17.1% Total 1.96 6, 36 100.0% 100.0%

As shown in Table. XIX and in Fig. 1, particle size distributions are different for both types of plecanatide. During the analysis, it was observed that the precipitated plecanatide contained some larger particles that could be easily destroyed. It was also noted that the lyophilized plecanatide appeared to be flaky and adhered to the top and bottom of the sieves, while no adhesion was observed for the precipitated plecanatide. This indicates the best processing properties of the precipitated plecanatide.

(4) Particle size and shape: FIG. 2 and 3 are optical microscopic analyzes of lyophilized and precipitated plecanatide samples, respectively. As seen in FIG. 2, the lyophilized plecanatide is in an amorphous form and has a non-uniform particle shape. They form physical aggregates with particles lying on top of each other. In FIG. 3 for precipitated plecanatide shows distinct individual particles. In terms of appearance and particle shapes, precipitated plecanatide may have better rheological properties and thus may facilitate processing of the solid during manufacture.

The lyophilized and precipitated forms of plecanatide show different physical appearance and properties in terms of density, particle size distribution and shape analyses. The precipitated form is more suitable for processing a solid dosage form during manufacture in terms of reduced dust generation, less buildup on processing equipment, and potentially less processing losses.

The precipitated form is more suitable for solid dosage form processing during manufacture (eg, low dose solid dosage form). The higher density of the deposited material may reduce the loss of aerosol or dust during weighing, transfer and mixing. A different particle shape has been shown to reduce the loss associated with sticking to screens or sieves. The higher density should improve the uniformity of the formulation because the particle size and density of the drug more closely matches the particle size and density of the excipients.

Example 7 Low dose formulation of precipitated SP-304 (as in WO2012/118972).

Plecanatide, purified by precipitation as described in Example 5, was processed further to obtain low dose formulations as described below.

Batch composition for dry blending.

Mixing.

Avicel PH 102 is screened through a 60 mesh sieve. V-blenders (1 quart (1.1 L), 4 quarts (4.4 L) and 16 quarts (17.6 L) are then powdered with sifted Avicel PH 102. SP-304 is sifted through a 200 mesh sieve and loaded into 1 quart (1.1 L) V-mixer. Approximately 80 g of Avicel PH 102 is then added to a 1 quart (1.1 L) mixer and the mixture is mixed for 10 minutes at 25 rpm. The mixture is then transferred to a 4 quart (4.4 L) V-mixer, which is dusted with sifted Avicel PH 102. 4 l). The rinse is repeated until all of the SP-304 is transferred to the 4 quart (4.4 L) mixer. Approximately 200 grams of Avicel is added to a 4 quart (4.4 L) mixer and the mixture is stirred for 10 minutes. The resulting mixture is then screened through a 60 mesh screen, transferred to a 16 quart (17.6 L) pre-powdered mixer (powdered 1500 g Avicel). A 4 quart (4.4 L) mixer was rinsed with Avicel and the rinsed material was transferred to a 16 quart (17.6 L) mixer. The remaining Avicel is added to a 16 quart (17.6 L) mixer and the mixture is stirred for 10 minutes. received

- 61 042294 the mixture is passed through a cone mill, washed with excess Avicel, and then returned to the mixer for 16 quarts (17.6 l) and additionally mixed for 5 minutes. The appropriate amount of magnesium stearate is weighed, screened through a 60 mesh sieve, and added to a 16 quart (17.6 L) blender. The resulting mixture was stirred for 2 minutes. The resulting mixture is then either packed into capsules or compressed to form tablets.

Encapsulation.

The MG2 Planeta capsule filler is installed. Determine the average weight of the empty shells of the capsules and calculate the target weight of the filling of the capsules (±5%). The mixture after the above process is added to the capsule filler bin and encapsulation is started. The operating mass parameters are adjusted manually. The resulting capsules are then sorted according to the target fill weight.

Pressing.

Install the Fette tablet press. Then the mixed mixture is loaded into the powder hopper and the tools are attached. The weight of each tablet is set to 100 mg±5% and the hardness to 4-6 Kp. The mass, hardness and thickness of the tablets are measured and recorded every 5-10 minutes. Abrasion measurements are also taken to ensure a satisfactory product.

Example 8 Synthesis and Purification of SP-304 by Solvent Exchange - Lyophilization.

Monocyclization of a linear crude peptide: First, a disulfide bond was formed between Cys ⁴ and Cys ¹² by oxidation of H ₂ O ₂ at pH 8.0-8.5 to obtain a monocyclized crude peptide. The linear crude peptide was added slowly and dissolved in 0.5% ammonium acetate buffer in water pre-added with H ₂ O ₂ at a ratio of 100:9 grams of peptide to ml H ₂ O ₂ to give a final concentration of the crude product of approximately 1.0 mg/ml . The pH of the solution was then adjusted to 8.5 with NH4OH solution while stirring without a lid. The oxidative monocyclization reaction was monitored using HPLC method 2. When the area % of the linear crude peptide was <5.0% of the area % of the monocyclized peptide, the oxidation reaction was stopped by adjusting the pH of the peptide solution to 1.7-2 using TFA solution. The peptide solution was then transferred to the next step to form a dicyclized peptide.

Dicyclization of the monocyclized peptide.

A disulfide bond between Cys7 and Cys15 was formed with 3.0% (w/v) I ₂ in acetonitrile solution. The disulfide bridge was generated while simultaneously removing the Acm side chain protecting groups present on the remaining Cys residues. The oxidative dicyclization reaction was monitored using HPLC method 2. Excess iodine was inactivated with a solution of 0.1 M ascorbic acid in water. After completion of the reaction, the pH of the dicyclized peptide was adjusted to 6.5-7 using a solution of NH ₄ OH, and received material for primary purification.

Primary Purification of the Dicyclized Crude Peptide: Then, a solution of the dicyclized crude peptide obtained from the oxidation steps was applied to a preparative RP-HPLC column packed with C18 Reverse Phase Chromatography Resin, controlled by a preparative HPLC system. The peptide was eluted in 1% triethylamine phosphate (TEAP) in water, pH7/ACN buffer. HPLC method 1 was used to clarify the percentage purity of fractions and pools obtained during the primary purification.

Purification of the recycled dicyclized peptide pool(s): After primary purification, fractions requiring further purification were purified based on the purity of the fraction pools using 1%TEAP in a buffer system of water, pH7,/ACN or 0.2% acetic acid in water /acn. Then, the dicyclized crude peptide solution obtained from the oxidation steps was applied to a preparative RP-HPLC column packed with C18 reverse phase chromatography resin controlled by a preparative HPLC system. The peptide was eluted in 1% TEAP in water, pH7/ACN buffer. HPLC method 1 was used to clarify the percentage purity of fractions and pools obtained during the re-purification.

Secondary purification and salt exchange: After secondary purification, fractions requiring further purification were purified based on the purity of fraction pools using 1% TEAP in water, pH7/ACN or 0.2% acetic acid in water/ACN buffer system. HPLC method 1 was used to clarify the percentage purity of fractions and pools obtained during the secondary purification. SELC method 1 was used to refine the percentage purity of the main pool obtained during the entire purification run before proceeding to the next step.

Solvent exchange: Material meeting the criteria of the main pool was loaded onto a flanged preparative RP-HPLC column in the reverse direction, washed with a 99:1 water to isopropanol solution in the reverse direction, and eluted with a 40:60 isopropanol:water solution in the forward direction. direction. The collected peptide solution was tested by HPLC method 1 to ensure purity, then the peptide solution was filtered, rotary evaporated to remove excess isopropanol to less than 5%, followed by partial lyophilization, e.g., for at least 96 hours.

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Dilution and Final Lyophilization: The partially lyophilized dry peptide was diluted in water to obtain a homogeneous batch. An amount of 0.5% (w/w) ammonium acetate based on dry peptide was added to the solution and mixed until the ammonium acetate/peptide dissolved. The material was subjected to analysis by SELC method 1 to check the purity. The dissolved material was placed in a lyophilizer tray and kept under vacuum for at least 120 hours to obtain the final dry peptide material.

Methods of control during the technological process.

HPLC Method 1: The following RP-HPLC analytical method can be used to verify HPLC percentage purity after the primary and secondary purification steps, or to document a specified batch.

HPLC parameters.

Column: Waters SunFire™ C8, 3.5 µm, 4.6x150 mm or equivalent.

Wavelength: 215 nm.

Mobile phase A: 0.02 M TEAP in water, pH 6.5 (BA assay buffer).

Mobile phase B: 100% ACN.

Injection volume: 5-50 µl.

Column temperature: 40°C.

Gradient elution parameters.

HPLC Method 2: The following RP-HPLC analytical method can be used to refine the percent HPLC purity of the linear crude peptide and to monitor the progress of the oxidative cyclization steps.

HPLC parameters.

Column: Kromasil C18 5 µm 100A 4.5x250mm or equivalent.

Wavelength: 215 nm.

Mobile phase A: 0.02 M TEAP in water, pH 6.5 (BA assay buffer).

Mobile phase B: 100% ACN.

Injection volume: 5-50 µl.

Column temperature: 40°C.

Gradient elution parameters.

SELC method 1: The following RP-HPLC analytical method can be used to clarify the percentage purity of the main pool and the material of the final peptide product before lyophilization.

SELC parameters.

Column: Waters Acquity BEN C18, 1.7 µm, 150x2.1 mm, P/N 186002353 with Waters Acquity 0.2 µm filter unit built into the column, P/N 205000343.

Wavelength: 215 nm.

Mobile phase A: 48/52/0.16 - MeOH/water/TFA.

Injection volume: 5 µl.

Column temperature: 10°C.

Isocratic elution parameters.

Two batches were made in the manner described in the example: batch #121026 IPA2 with a batch size of 50 g and batch #121026 B with a batch size of 930 g. As illustrated in FIG. 4, the peptide of batch #121026 IPA2 had a HPLC chromatographic purity of ~98%. Other characteristics of the two parties are summarized in table. XX is lower compared to batches made by the methods described in WO2012/118972.

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Table XX

ACA Lyophilization Process (Average or Range) Lyophilization process with ACA (batch 101221) WO 2012/118972 Lyophilization process with ACA (batch 110425) WO 2012/118972 Deposition process (batch Feb2012) WO 2012/118972 precipitation Process (batch 120210) WO 2012/118972 Deposition process (batch 130117) Solvent Change Process (Batch 121026 IPA2) Solvent Change Process (Lot 121026 B) Lot size 1110-2580 1142 1110 g 0.5 g 10 g 200 g 50 g 930 g Acetamide 88-453ppm 350ppm 356ppm <28ppm <47ppm 35ppm <18ppm TFA <0.040.25% 0.2% 0.14% 0.09% 0.10% <0.04% <0.04% Acetate 0.6-4.7% <0.6% 0.6% <0.01% 0.08% 0.21% Ammonium 0.02-1.94% 1.05% 1.58% 0.23% 0.61% 0.17% 0.11% Degradation product (RRT 1.33) <0.10- 0.15% <0.10% 0.10% 0.31% 0.89% 0.10% 0.11% Chromatography High HPLC purity 97.3% 97.6% 97.0% 97.8% 97.1% total impurities 2.24% (n=7) 1.46-2.71% 2.33% 1.87% 2.51% 2.89% 2.17% 2.92%

ROS (residual organic solvent) ACN 0-40 ppt Total <20ppm ACN 40ppm Ethyl acetate 20 PPT IPA 27000ppm Methanol 250 ppt MTBE 20 ppm Tetrahydrofura n 70 ppm IPA 90,000 PPm IPA 1700ppm ACN 240ppm IPA 320ppm Bulk density 0.03-0.06 g / cm ³ 0.033 g/ ^cm3 0.486 g/ ^cm3 0.64 g/ ^cm3 0.03 g/ ^cm3 0.056 g/ ^cm3 Air dispersant particle size (D50 µm) 182-234 235 300 635 91 Liquid Dispersant Particle Size (µm) D10:8 D50:22 D90:37 D10:7 D50:21 D90:59 D10:8 D50:21 D90:40 D10:4 D50:17 D90:34 x-ray Amorphous Amorphous Amorphous Amorphous Sieve analysis (remaining percentage) 600 µm 3.6% 425 µm 13.3% 250 µm 8.7% 150 µm 26.0% 106 µm 33.2% 600 µm 7.1% 425 µm 2.7% 250 µm 10.5% 150 µm 32.7% 106 µm 19.2% 600 µm 11.0% 425 µm 13.1% 250 µm 24.7% 150 µm 23.7% 106 µm 11.0% 600 µm 42.1% 425 µm 26.3% 250 µm 21.1% 150 µm 7.9% 106 µm 2.6% 600 µm 3.6% 425 µm 7.1% 250 µm 17.9% 150 µm 10.7% 10 6 µm 2 5 % Water 5.4% 7.3% 4.9% 5.3% 2.7%

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Claims (12)

Table XXI. Plecanatide Drug Substance Stability, Lot 121026B, Storage at 25°C Lot No.: 121026B Container: 60 ml transparent bottles Test T=0 3 months 6 months Appearance (visual) White powder White powder White powder Identity by SELC I Corresponds Corresponds Corresponding tvuet Chromatographic purity of plecanatide (SELC) (% area) 97.1% 97.0% 96.4% total impurities 2.92% 2.90% 3.61% Water content (KF) 2.7% 8.7% 8.3% CLAIM 1. A purified peptide preparation containing a peptide containing a GCC agonist amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 9 and 104, wherein the purified peptide preparation has less than 0.05% trifluoroacetic acid (TFA) based on the total weight of the peptide in a peptide preparation, wherein the purified peptide preparation is obtained by a method including: the presence of a first peptide solution containing water, acetonitrile and a peptide containing the amino acid sequence of a GCC agonist selected from the group consisting of SEQ ID NOs: 1, 9 and 104; loading the column with C18 or polymer adsorbent with the first peptide solution to adsorb the peptide on the column with C18 or polymer adsorbent, elution of the peptide from the column with C18 or polymer adsorbent using an aqueous solution of isopropanol to obtain a second peptide solution, reducing the amount of isopropanol in the second peptide solution, lyophilization of the second peptide solution so that a dry peptide is obtained, dissolving the dry peptide in an aqueous ammonium acetate solution to obtain a third peptide solution, and lyophilizing the third peptide solution so that a purified peptide is obtained. 2. Purified peptide preparation according to claim 1, wherein the peptide is stable at 25°C for at least three months. 3. Purified peptide preparation according to claim 1, where the peptide has a particle size distribution that is different: a) a D10 value of approximately 2-15 µm; b) a D50 value of approximately 15-50 µm; And c) a D90 value of approximately 40-80 µm as measured by light scattering with a liquid dispersant. 4. Purified peptide preparation according to claim 1, wherein the peptide has a bulk density of not more than 0.06 g/ml. 5. Purified peptide preparation according to claim 1 or 2, wherein the peptide has a chromatographic purity of at least 95%. 6. A purified peptide preparation according to any one of claims 1 to 5, wherein the water content of the peptide preparation is less than 10% of the total weight of the peptide in the peptide preparation. 7. A purified peptide preparation according to any one of claims 1 to 6, wherein the peptide preparation also contains less than 0.5% by weight relative to the weight of the peptide of one or more impurities selected from the group consisting of acetonitrile, alcohols, ammonium, and acetate. 8. Purified peptide preparation according to claim 7, wherein the peptide preparation contains less than 300 ppm of acetonitrile. 9. A purified peptide preparation according to any one of claims 7-8, wherein the peptide preparation contains less than 1000 ppm of isopropanol. 10. A purified peptide preparation according to any one of claims 7-9, wherein the peptide preparation contains less than 600 ppm of isopropanol. 11. A purified peptide preparation according to any one of claims 7-10, wherein the peptide contains less than 0.25% acetate based on the total weight of the peptide in the peptide preparation. 12. A purified peptide preparation according to any one of claims 1 to 11, wherein the peptide preparation further comprises less than 2% of peptide topoisomers based on the total weight of the peptide in the peptide preparation. -