Ultra-pure agonists of guanylate cyclase c, method of making and using same

RELATED APPLICATIONS

0001This application claims priority to and benefit of provisional application <patcit id="pcit0001" dnum="US61831402"><text>USSN 61/831,402 filed on June 5, 2013</text></patcit>, the contents of which are herein incorporated by reference in their entireties.

FIELD OF THE INVENTION

0002The present invention relates to processes of purifying guanylate cyclase C peptide agonists useful for preparing formulations for the treatment and prevention of various diseases and disorders.

BACKGROUND OF THE INVENTION

0003Guanylate cyclase C is a transmembrane form of guanylate cyclase that is expressed on various cells, including gastrointestinal epithelial cells (reviewed in <nplcit id="ncit0001" npl-type="s"><text>Vaandrager 2002 Mol. Cell. Biochem. 230:73-83</text></nplcit>). It was originally discovered as the intestinal receptor for the heat-stable toxin (ST) peptides secreted by enteric bacteria and which cause diarrhea. The ST peptides share a similar primary amino acid structure with two peptides isolated from intestinal mucosa and urine, guanylin and uroguanylin (<nplcit id="ncit0002" npl-type="s"><text>Currie, et al., Proc. Nat'l Acad. Sci. USA 89:947-951 (1992</text></nplcit>); <nplcit id="ncit0003" npl-type="s"><text>Harnra, et al., Proc. Nat'l Acad. Sci. USA 90:10464-10468 (1993</text></nplcit>); <nplcit id="ncit0004" npl-type="s"><text>Forte, L., Reg. Pept. 81:25-39 (1999</text></nplcit>); <nplcit id="ncit0005" npl-type="s"><text>Schulz, et al., Cell 63:941-948 (1990</text></nplcit>); <nplcit id="ncit0006" npl-type="s"><text>Guba, et al., Gastroenterology 111:1558-1568 (1996</text></nplcit>); <nplcit id="ncit0007" npl-type="s"><text>Joo, et al., Am. J. Physiol. 274:G633-G644 (1998</text></nplcit>)).

0004In the intestines, guanylin and uroguanylin act as regulators of fluid and electrolyte balance. In response to high oral salt intake, these peptides are released into the intestinal lumen where they bind to guanylate cyclase C localized on the luminal membrane of enterocytes (simple columnar epithelial cells of the small intestines and colon). The binding of the guanylin peptides to guanylate cyclase C induces electrolyte and water excretion into the intestinal lumen via a complex intracellular signaling cascade that is initiated by an increase in cyclic guanosine monophosphate (cGMP).

0005The cGMP-mediated signaling that is initiated by the guanylin peptides is critical for the normal functioning of the gut. Any abnormality in this process could lead to gastrointestinal disorders such as irritable bowel syndrome (IBS) and inflammatory bowel diseases. Inflammatory bowel disease is a general name given to a group of disorders that cause the intestines to become inflamed, characterized by red and swollen 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 sections of the gastrointestinal tract. Ulcerative colitis is exclusively an inflammatory disease of the colon, the large intestine. Unlike Crohn's disease, in which all layers of the intestine are involved, and in which there can be normal healthy bowel in between patches of diseased, bowel, ulcerative colitis affects only the innermost lining (mucosa) of the colon in a continuous manner. Depending on which portion of the gastrointestinal tract is involved, Crohn's disease may be referred to as ileitis, regional enteritis, colitis, etc. Crohn's disease and ulcerative colitis differ from spastic colon or irritable bowel syndrome, which are motility disorders of the gastrointestinal tract. Gastrointestinal inflammation can be a chronic condition. It is estimated that as many as 1,000,000 Americans are afflicted with inflammatory bowel disease, with male and female patients appearing to be equally affected. Most cases are diagnosed before age 30, but the disease can occur in the sixth, seventh, and later decades of life.

0006IBS and chronic idiopathic constipation are pathological conditions that can cause a great deal of intestinal discomfort and distress but unlike the inflammatory bowel diseases, IBS does not cause the serious inflammation or changes in bowel tissue and it is not thought to increase the risk of colorectal cancer. In the past, inflammatory bowel disease, celiac disease, and IBS were regarded as completely separate disorders. Now, with the description of inflammation, albeit low-grade, in IBS, and of symptom overlap between IBS and celiac disease, this contention has come under question. Acute bacterial gastroenteritis is the strongest risk factor identified to date for the subsequent development of post infective irritable bowel syndrome. Clinical risk factors include prolonged acute illtiess and the absence of vomiting. A genetically determined susceptibility to inflammatory stimuli may also be a risk factor for irritable bowel syndrome. The underlying pathophysiology indicates increased intestinal permeability and low-grade 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 major role in pathophysiology of IBS. The activity of 5-HT is regulated by cGMP.

0007While the precise causes of IBS and inflammatory bowel diseases (IBD) are not known, a disruption in the process of continual renewal of the gastrointestinal mucosa may contribute to disease pathology in IBD and aggravate IBS. The renewal process of the gastrointestinal lining is an efficient and dynamic process involving the continual proliferation and replenishment of unwanted damaged cells. Proliferation rates of cells lining the gastrointestinal mucosa are very high, second only to the hematopoietic system. Gastrointestinal homeostasis depends on both the proliferation and programmed cellular death (apoptosis) of epithelial cells lining the gut mucosa. Cells are continually lost from the villus into the lumen of the gut and are replenished at a substantially equal rate by the proliferation of cells in the crypts, followed by their upward movement to the villus. The rates of cell proliferation and apoptosis in the gut epithelium can be increased or decreased in a variety of circumstances, <i>e.g</i>., in response to physio logical stimuli such as aging, inflammatory signals, hormones, peptides, growth factors, chemicals and dietary habits. In addition, an enhanced proliferation rate is frequently associated with a reduction in turnover time and an expansion of the proliferative zone. The proliferation index is much higher in pathological states such as ulcerative colitis and other gastrointestinal disorders. Intestinal hyperplasia is a major promoter of gastrointestinal inflammation. Apoptosis and cell proliferation together regulate cell number and determine the proliferation index. Reduced rates of apoptosis are often associated with abnormal growth, inflammation, and neoplastic transformation. Thus, both increased proliferation and/or reduced cell death may increase the proliferation index of intestinal tissue, which may in turn lead to gastrointestinal inflammatory diseases.

0008In addition to a role for uroguanylin and guanylin as modulators of intestinal fluid and ion secretion, these peptides may also be involved in the continual renewal of gastrointestinal mucosa by maintaining the balance between proliferation and apoptosis. For example, uroguanylin and guanylin peptides appear to promote apoptosis by controlling cellular ion flux. Given the prevalence of inflammatory conditions in Western societies a need exists to improve the treatment options for inflammatory conditions, particularly of the gastrointestinal tract.

0009Peptide agonists of guanylate cyclase C agonists ("GCC agonists") are described in <patcit id="pcit0002" dnum="US7041786B"><text>U.S. Patent Nos. 7,041,786</text></patcit>, <patcit id="pcit0003" dnum="US7799897B"><text>7,799,897</text></patcit>, and U.S. Patent Application Publication Nos. <patcit id="pcit0004" dnum="US20090048175A"><text>US2009/0048175</text></patcit>, <patcit id="pcit0005" dnum="US20100069306A"><text>US 2010/0069306</text></patcit>, <patcit id="pcit0006" dnum="US20100120694A"><text>US 2010/0120694</text></patcit>, <patcit id="pcit0007" dnum="US20100093635A"><text>US 2010/0093635</text></patcit>, and <patcit id="pcit0008" dnum="US20100221329A"><text>US 2010/0221329</text></patcit>, and <patcit id="pcit0009" dnum="WO2012118972A"><text>WO2012/118972</text></patcit>. However, the previous syntheses of peptides for pharmaceutical application present a number of special problems such as an overall low yield (e.g., less than 10%), and/or high levels of impurities (e.g., contaminants resulted from organic solvents used during syntheses or purification, and degradation products or topoisomers created, e.g., during purification).

SUMMARY OF THE INVENTION

0010In one aspect, the present invention is derived from the effort to solve various unexpected problems encountered during the purification processes of peptide GCC agonists for pharmaceutical application, such as the lyophilization process and precipitation process described in <patcit id="pcit0010" dnum="WO2012118972A"><text>WO2012/118972</text></patcit>. The methods described herein provide a solution to those problems.

0011In one aspect, the present invention provides a purified peptide comprising the GCC agonist sequence selected from the group consisting of SEQ ID NOs: 1, 9, and 104, wherein the purified peptide has the following characteristics: <ol id="ol0001" compact="compact"><li>a) has a bulk density of not greater than 0.1 g/mL;</li><li>b) contains less than 50 ppm acetamide;</li><li>c) less than 0.3% alpha-Asp-9-plenacanatide.</li></ol>

0012The purified peptide can have one or more of the following features.

0013For, example, the peptide is stable at 25°C for at least three months.

0014For example , the peptide has a particle size distribution having a D10 value of between about 2 tp 15 µm;a D50 value of between about 15-50 µm; and a D90 value of between about 40-80 µm when measured by light scattering with liquid dispersant.

0015For example, the purified peptide contains no more than 35 ppm acetamide (e.g., ≤ 18 ppm).

0016For example, the purified peptide contains less than 0.15% alpha-Asp-9-plenacanatide (which has a Relative Retention Time (RRT) of ~1.33 from the ultra-performance liquid chromatography (UPLC) analysis described herein).

0017For example, the purified peptide has a bulk density of not greater than 0.09 g/mL, not greater than 0.08 g/mL, not greater than 0.07 g/mL, not greater than 0.06 g/mL, not greater than 0.05 g/mL, not greater than 0.04 g/mL, or not greater than 0.03 g/mL.

0018For example, the purified peptide is substantially free of water (e.g., water content not exceeding 10%, 9%, 8%,7%. 6%, 5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.5%, 0.25%, or 0.1%, of the total weight of the peptide).

0019For example, the purified peptide has a chromatographic purity of no less than 95%, no less than 96%, or no less than 97%.

0020For example, the total content of impurities in the purified peptide is less than 3% (e.g., &lt;2% or &lt;1%).

0021For example, the purified peptide is further substantially free of one ormore impurities selected from acetonitrile, alcohols, ammonium, acetates, and TFA.

0022For example, the purified peptide contains less than 300 ppm acetonitrile (e.g., &lt; 250 ppm).

0023For example, the purified peptide contains less than 0.2% TFA (e.g., &lt;0.15%, &lt;0.1%, &lt;400 ppm, &lt; 300 ppm, &lt; 200 ppm, &lt; 100 ppm, or &lt; 50 ppm).

0024For example, the purified peptide contains less than 0.2% isopropanol, i.e., IPA (e.g., &lt;0.15%, &lt; 0.1%, &lt; 1000 ppm, &lt; 900 ppm&lt; 800 ppm, &lt; 700 ppm, &lt; 600 ppm, &lt; 500 ppm, &lt; 400 ppm, &lt; 300 ppm, &lt; 200 ppm, &lt; 100 ppm, &lt; 50 ppm, or &lt; 20 ppm).

0025For example, the purified peptide contains less than 0.25 % acetate (e.g., &lt;0.2% or &lt;0.1%).

0026For example, the purified peptide is substantially free of topoisomers (e.g., &lt;0.4%, &lt;0.3%, &lt;0.2% or &lt;0.1%).

0027For example, the purified peptide is substantially free of iso-Asp2-plecanatide (RRT 0.96-0.97) (e.g., &lt;0.4%, &lt;0.3%, &lt;0.2% or &lt;0.1%).

0028In another aspect, the invention also provides a process of purifying/isolating the peptide comprising the GCC agonist sequence selected from the group consisting of SEQ ID NOs: 1-251. The process includes: <ul id="ul0001" list-style="none" compact="compact"><li>providing a first peptide solution comprising a peptide comprising the GCC agonist sequence selected from the group consisting of SEQ ID NOs: 1-251, water, and acetonitrile;</li><li>loading a C18 or polymeric adsorbent column with the first peptide solution to adsorb the peptide onto the polymeric adsorbent column,</li><li>eluting the peptide off the C18 or polymeric adsorbent column with an alcohol aqueous solution to form a second peptide solution,</li><li>reducing the amount of alcohol in the second peptide solution, and</li><li>lyophilizing the second peptide solution such that a dry peptide is obtained.</li></ul>

0029The process can include one or more of the following features.

0030For example, the alcohol aqueous solution comprises isopropanol (e.g., with isopropanol content in the alcohol aqueous solution being about 40%).

0031In another embodiment, the alcohol aqueous solution comprises propanol, tert-butanol, 2-butanol, or ethanol.

0032For example, the first peptide solution further comprises acetamide.

0033For example, the first peptide solution further comprises acetic acid (e.g., 0.2%) or triethylamine phosphate (e.g., 1%).

0034For example, the amount of alcohol (e.g., isopropanol) in the second peptide solution is reduced e.g. by rotoevaporation to less than 5%.

0035For example, the process further comprises dissolving the dry peptide in water to form a third peptide solution after lyophilization. For example, the third peptide solution further comprises ammonium acetate or ammonium hydroxide (e.g., such that the third solution has a pH value of about 5).

0036For example, the process further comprises lyophilizing the third peptide solution such that a purified peptide is obtained.

0037For example, the peptide in the first peptide solution is prepared by the fragment condensation process (i.e., hybrid solution- and solid-phase process) as described in <patcit id="pcit0011" dnum="WO2012118972A"><text>WO2012/118972</text></patcit>. In one embodiment, the first peptide solution is obtained from a salt exchanging step in which the peptide is washed with an aqueous acetonitrile solution comprising triethylamine phosphate or acetic acid.

0038For example, the polymeric adsorbent column is a preparative C₁₈ RP-HPLC column. In one embodiment, the polymeric adsorbent column comprises a polystyrene resin. In particular, the resin is selected so that the purified peptide eluted or desorbed is not less than 80% of the peptide amount adsorbed on the resin, e.g., not less than 85%, not less than 90%, or not less than 95%. In one embodiment, the resin is formed of crosslinked polystyrene with an average pore diameter greater than 5 nm, e.g., about 6-8 nm, 10-15 nm, 15-20 nm, or 25-30 nm.

0039In yet another aspect, the invention also provides a purified peptide prepared by the purification process of the invention. The purified peptides may have one or more of the following features.

0040For example, the purified peptide comprises the GCC agonist sequence selected from the group consisting of SEQ ID NOs: 1, 9, and 104.

0041For example, the purified peptide has a chromatographic purity of no less than 96%, no less than 97%, or no less than 98%. For example, the GCC agonist peptide has chromatographic impurity content of no greater than 4%, no greater than 3.5%, no greater than 3%, no greater than 2.5%), no greater than 2%, no greater than 1.5%, or no greater than 1%. The chromatographic impurity content is determined as total area percentages of impurities by HPLC. The chromatographic impurity content includes topoisomer content. The impurities do not include any pharmaceutically acceptable excipient used for drug formulation.

0042For example, the purified peptide is substantially free of contaminants resulted 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 contaminant content 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 weight of the peptide. For example, the purified peptide contains &lt; 50 ppm acetamide (e.g., ≤ 35 ppm or ≤ 18 ppm), &lt; 300 ppm acetonitrile (e.g., &lt; 250 ppm), &lt; 1000 ppm TFA (e.g., &lt; 400 ppm, &lt; 300 ppm, &lt; 200 ppm, &lt; 100 ppm, or &lt; 50 ppm), &lt;2000 ppm isopropanol (e.g., &lt;1500 ppm, &lt;1000 ppm, &lt;500 ppm, &lt; 400 ppm, &lt; 300 ppm, &lt; 200 ppm, &lt;: 100 ppm, &lt; 50 ppm, or ≤ 20 ppm), , and/or &lt; 0.25 % acetate (e.g., &lt;0.2% or &lt;0.1%). The content of contaminants can be determined by conventional methods such as gas chromatography. Preferably, the residual solvents in the purified peptide of the invention are less than the limits set in the ICH guidelines, e.g., IMPURITIES: GUIDELINE FOR RESIDUAL SOLVENTS Q3C(R5) (available at http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q3C/Step4/Q 3C_R5_Step4.pdf).

0043For example, the purified peptide contains less than 0.3% (e.g., &lt; 0.15%) alpha-Asp-9-plenacanatide (RRT 1.33).

0044For example, the purified peptide has a bulk density of not greater than 0.09 g/mL, not greater than 0.08 g/mL, not greater than 0.07 g/mL, not greater than 0.06 g/mL, not greater than 0.05 g/mL, not greater than 0.04 g/mL, or not greater than 0.03 g/mL.

0045For example, the purified peptide is substantially free of iso-Asp2-plecanatide (RRT ~ 0.96-0.97). In this context "substantially" free of iso-Asp2-plecanatide means that the iso-Asp2-plecanatide 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 weight of the peptide.

0046For 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 weight of the peptide.

0047For example, 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%, of the total weight of the peptide.

0048For example , the peptide has a particle size distribution having a D10 value of between about 2 tp 15 µm;a D50 value of between about 15-50 µm; and a D90 value of between about 40-80 µm when measured by light scattering with liquid dispersant.

0049For example, the purified peptide has a particle size distribution characterized by a D50 value of about 600 µm when measured by light scattering with air dispersant. In comparison, the peptides purified from the lyophilization and precipitation processes described in <patcit id="pcit0012" dnum="WO2012118972A"><text>WO2012/118972</text></patcit> have D50 values of about 180-250 µm and about 300 µm, respectively.

0050For example, the purified peptide prepared by the processes of the invention has a suitable size distribution for pharmaceutical formulation. In one embodiment, the peptide (e.g., SP-304) has a size distribution (e.g., an average size of 80-120 µm) comparable to that of the pharmaceutical excipient (e.g., microcrystalline cellulose) used in the formulation, for example, in the 3 mg/day unit dose form. The size distribution of the purified peptide may vary based on the unit dose. For example, when unit dose is lower than 3 mg/day, the purified peptide in the pharmaceutical formulation has a smaller average size than that in the 3 mg/day dose. For example, the purified peptide prepared by the processes of the invention is milled to reach the suitable size distribution.

0051The size distribution of the peptide of the invention can be determined by traditional methods, such as sieve analysis, light obscuration or dynamic light scattering analyses.

0052The invention also relates to a formulation (e.g., an oral formulation) containing the peptides prepared and/or purified by the methods described herein and in particular, a low dose formulation containing 0.05-10 mg (e.g., 0.1 mg, 0.3 mg or 0.5 mg) of the purified peptides. The low-dose formulation can further have one or more additional features as described in <patcit id="pcit0013" dnum="WO201203780A"><text>WO2012/03780</text></patcit> and <patcit id="pcit0014" dnum="US20120237593A"><text>US 2012-0237593</text></patcit> and can be prepared by the methods disclosed therein, such as dry blending.

0053Other features and advantages of the invention will be apparent from and are encompassed by the following detailed description and claims.

BRIEF DESCRIPTION OF DRAWINGS

0054<ul id="ul0002" list-style="none"><li><figref idref="f0001">Figure 1</figref> is a graph showing particle size distribution by sieving analysis for lyophilized plecanatide and precipitated plecanatide.</li><li><figref idref="f0002">Figure 2</figref> is an optical microscopic image of lyophilized plecanatide.</li><li><figref idref="f0003">Figure 3</figref> is an optical microscopic image of precipitated plecanatide.</li><li><figref idref="f0004">Figure 4</figref> is a UPLC chromatogram of plecanatide isolated by one embodiment of the purification process of the invention.</li></ul>

DETAILED DESCRIPTION

0055In one aspect, the present invention provides a solution to various unexpected problems encountered during the purification processes of peptide GCC agonists for pharmaceutical application, such as the lyophilization process and precipitation process described in <patcit id="pcit0015" dnum="WO2012118972A"><text>WO2012/118972</text></patcit>.

0056In particular, it was found that the lyophilization process described in <patcit id="pcit0016" dnum="WO2012118972A"><text>WO2012/118972</text></patcit>, which involved use of acetonitrile/water solvent, unexpectedly resulted in enrichment of residual acetamide (a trace level impurity in acetonitrile) in the lyophilized plecanatide product with high acetamide content (i.e., ranging from 88 to 453 ppm among tested batches, or about 300 ppm on average), which hindered commercialization of plecanatide with doses higher than 3 mg/day. In addition, the lyophilized plecanatide product also had a high variability of residual salt levels such as TFA, acetate, and ammonium salts.

0057On the other hand, while the plecanatide product purified via the precipitation process described in <patcit id="pcit0017" dnum="WO2012118972A"><text>WO2012/118972</text></patcit> had low residual acetamide content (&lt;50 ppm) and higher bulk or tap density (i.e., about ten times higher) than the lyophilized product, the precipitated plecanatide product contained high levels of residual solvents (e.g., IPA as high as 90,000 ppm) due to difficulty in removing the solvents used for peptide precipitation. In addition, while low temperature heating (45 °C) during vacuum drying helped to reduce the residual isopropanol to the amount below the ICH limit of 5000 ppm (e.g., 1700 ppm), a thermal degradant, alpha-Asp-9-plecanatide, having a Relative Retention Time (RRT) of ~1.33 from ultra-performance liquid chromatography (UPLC) analysis, was then formed in the final peptide product at a concentration as high as 0.9 %. The content of the RRT 1.33 impurity also increases in plecanatide and tablets thereof during room-temperature storage. Therefore, the initial high level of this RRT 1.33 impurity in plecanatide produced by the precipitation process results in a smaller window of acceptance and hence shortens the usable shelf life of plecanatide drug products.

0058The purification processes of the invention solves the residual solvent problem and degradation problem by, for example, exchanging the acetonitrile/water solvent with IPA/water solvent followed by rotary evaporation to reduce IPA to a critical low level (e.g., &lt;5%), followed by a first sublot freeze drying, which resulted in low but unacceptable levels of residual solvents, then followed by reconstitution in water and final freeze drying resulting in low and acceptable levels of residual solvents for pharmaceutical application. In addition, the purification processes of the invention are suitable for scale up preparation/purification of peptides.

0059It was further unexpected discovered that adding a small amount of ammonium acetate buffer (e.g., 0.5% to dry peptide) during reconstitution with water (pH 5) prior to final lyophilization improves solubility of final product. Further, ammonium acetate buffer (pH 5) reduces the rate of plecanatide topo-isomerization in solution during lyophilization. The ammonium acetate buffer (pH 5) also controls residual salt levels in the final product with lower variability as compared to the lyophilization product described in <patcit id="pcit0018" dnum="WO2012118972A"><text>WO2012/118972</text></patcit>.

0060The peptide at the end of the purification or isolation processes of the invention has a low residual acetamide level (&lt;50 ppm) and a bulk density of not greater than 0.1 g/mL, as well as an overall lower level of residual solvents (e.g., &lt;500 ppm IPA, &lt;300 ppm ACN), low levels of degradation impurities (e.g., &lt;0.1 % alpha-Asp-9-plecanatide (RRT 1.33) degradant) and topoisomers (e.g., 0.4% or less), and low levels of residual salts (e.g., &lt;0.1% TFA, 0.08-0.23% acetate, and 0.11-0.17% ammonium). The comparison among plenacanatide products isolated by different methods is presented in Table XX <i>infra.</i>

0061The invention provides processes of purifying or isolating peptides, e.g., peptide GCC agonists, in particular peptides prepared via a hybrid solution- and solid-phase process, e.g., as described in <patcit id="pcit0019" dnum="WO2012118972A"><text>WO2012/11 8972</text></patcit>, which is hereby incorporated by reference in its entirety. The hybrid process includes providing two or more fragments of a peptide of interest via solid-phase and/or solution-phase syntheses and coupling them via a solution-phase synthesis to obtain the target peptide. The process may further include, if needed, oxidative cyclization of cysteine amino acid residues of a linear peptide formed by the fragment coupling to produce a cyclized peptide.

0062The fragments described above can be prepared by standard solution phase peptide synthesis or solid phase peptide synthesis techniques in which a peptide linkage occurs through the direct condensation of the amino group (i.e., NH₂) of a first amino acid with the carboxy group (i.e., COOH) of a second amino acid with the elimination of a water molecule. In one embodiment, at least one of the fragments is prepared by solid phase peptide synthesis.

0063Peptide bond synthesis by direct condensation, as formulated above, requires suppression of the reactive character of the amino group of the first and of the carboxyl group of the second amino acid. The masking substituents (i.e., protecting groups) must permit their ready removal, without inducing breakdown 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 its constituting amino acids, are masked by protecting groups, unless otherwise specified. The term "deprotected peptide" or "deprotected peptide fragment" refers to a peptide or peptide fragment, in which all reactive groups on its constituting amino acids, are free from being masked by protecting groups, unless otherwise specified. The term "reactive groups" refers to the groups forming the peptide bond and those interfering with the peptide bond formation, such as amino, carboxyl, hydroxyl, and thiol (as in cysteine) groups. Examples of protecting groups for amino include and are not limited to 9-fluorenylmethyloxycarbonyl (Fmoc), tert-butoxycarbonyl (Boe), benzoyl (Bz), acetyl (Ac), and benzyl (Bn). Examples of protecting groups for carboxyl include trityl (triphenylmethyl, Trt) and O-tert-butyl (OtBu). Examples of protecting groups for thiol include acetamidomethyl (Acm), tert-butyl (tBu), 3-nitro-2-pyridine sulfenyl (NPYS), 2-pyridine-sulfenyl (Pyr), and trityl (Trt). Additional examples of protecting groups are described in <nplcit id="ncit0008" npl-type="b"><text>Greene, T.W., Wuts, P.G. M., Protective Groups in Organic Synthesis, 3rd edition, John Wiley &amp; Sons: New York, 1999</text></nplcit>, whose context is incorporated by reference herein.

0064For example, the hybrid synthetic methods have been used for preparing SP-304 (plecanatide). In particular, three peptide fragments, A, B and C are prepared and then a linear peptide sequence is assembled by the condensation of fragment A, B and C as follows: preparing fragment A, Boe-Asn(Trt)-Asp(OtBu)-Glu(OtBu)-Cys(Trt)-Glu(OtBu)-Leu-OH, by solid phase from 2-chloro-trityl chloride resin; preparing fragment B, Fmoc-Cys(Acm)-Val-Asn-Val-Ala-Cys(Trt)-Thr(tBu)-Gly-OH, by solid phase from 2-chlorotrityl chloride resin; preparing fragment C, Cys(Acm)-Leu-OtBu, by solution phase synthesis, coupling fragments B and C in solution phase to yield fragment B-C, and coupling fragments A and B-C to yield linear peptide A-B-C.

0065The side-chain-protected Fragments A (BocAA1-6OH) and B (FmocAA7-14OH) can be prepared by Fmoc SPPS using the super acid-sensitive 2-chlorotrityl chloride (2-ClTrt) resin and Fmoc-protected amino acid derivatives, as shown in Scheme 1 below. <chemistry id="chem0001" num="0001"><img file="EP4424697A2_D0001.tif" /></chemistry><chemistry id="chem0002" num="0002"><img file="EP4424697A2_D0002.tif" /></chemistry>

0066Fragment C (HAA15-16OtBu) can be prepared by the solution phase synthesis and then be coupled to Fragment B (FmocAA7-14OH) in solution phase to give Fragment B-C (FmocAA7-16OtBu). The Fmoc protecting group can then be removed from Fragment B-C (FmocAA7-16OtBu) to give BAA7-16OtBu, which is then coupled to Fragment A (BocAA1-6OH) to yield side-chain-protected linear SP-304 (BocAA1-16OtBu), as shown in Scheme 2 below. <chemistry id="chem0003" num="0003"><img file="EP4424697A2_D0003.tif" /></chemistry><chemistry id="chem0004" num="0004"><img file="EP4424697A2_D0004.tif" /></chemistry>

0067The side-chain-protected linear SP-304 (BocAA1-16OtBu) can be treated with trifluoroacetic acid/triisopropylsilane/ethanedithiol (TFA/TIS/EDT) to give the partially protected SP-304 (HAA1-16OH) in which the 2 S-Acm groups (as shown in Scheme 2 above) are intact. The partially protected linear SP-304 (HAA1-16OH) can be oxidized by H₂O₂, 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. <chemistry id="chem0005" num="0005"><img file="EP4424697A2_D0005.tif" /></chemistry><chemistry id="chem0006" num="0006"><img file="EP4424697A2_D0006.tif" /></chemistry>

0068The solution of crude dicyclic SP-304 can then be purified and concentrated, as shown in Scheme 3 above, by loading the solution to a polystyrenic adsorbent resin (e.g., D101 (Anhui Sanxing (China); crosslinked polystyrene; surface area 500-550 rn²/g; average pore diameter: 9-10 nm, pore volume: 1.18-1.24 ml/g; bulk density: 0.65-0.70 g/ml; specific density: 1.03-1.07 g/ml; moisture: 67-75%; particle size: 0.315~1.2.5 mm ≧95%; effective diameter: 0.4~0.7 mm; uniformity coefficient: ≤1.6%), DA201C, DA201H, ADS-8, and ADS-5) column, eluting the dicyclic SP-304 from the column with an eluent (e.g., a 90% ethanol aqueous solution), concentrating the collected SP-304 solution under reduced pressure, and precipitating SP-304 with methyl t-butyl ether (MTBE). The precipitate can then be collected by filtration or centrifugation, dried under high vacuum to give SP-304 in solid form.

0069As illustrated in Scheme 4 above, the solution of crude dicyclic SP-304 can also be purified directly on preparative HPLC C18 column with acetonitrile (ACN), methanol, and/or water in various buffer systems. The crude dicyclic SP-304 can also be purified via other methods known to a skilled person in the art.

0070Those of ordinary skill in the art will recognize that, in solid phase synthesis, deprotection, and coupling reactions must go to completion and the side-chain blocking groups must be stable throughout the synthesis.

0071Acetylation of the N-terminal can be accomplished by reacting the final peptide with acetic anhydride before cleavage from the resin. C-amidation is accomplished using an appropriate resin such as methylbenzhydrylamine resin using the Boc technology.

0072In solution phase synthesis, a wide variety of coupling methods and protecting groups may be used (<i>See,</i><nplcit id="ncit0009" npl-type="b"><text>Gross and Meienhofer, eds., "The Peptides: Analysis, Synthesis, Biology," Vol. 1-4 (Academic Press, 1979</text></nplcit>); <nplcit id="ncit0010" npl-type="b"><text>Bodansky and Bodansky, "The Practice of Peptide Synthesis," 2d ed. (Springer Verlag, 1994</text></nplcit>)). In addition, intermediate purification and linear scale up are possible. Those of ordinary skill in the art will appreciate that solution synthesis requires consideration of main chain and side chain protecting groups and activation method. In addition, careful fragment selection is necessary to minimize racemization during fragment condensation. For example, racemization is minimized when fragments contain C-terminal Gly or Pro. Solubility considerations are also a factor. Solid phase peptide synthesis uses an insoluble polymer for support during organic synthesis. The polymer-supported peptide chain permits the use of simple washing and filtration steps instead of laborious purifications at intermediate steps. Solid-phase peptide synthesis may generally be performed according to the method of<nplcit id="ncit0011" npl-type="s"><text> Merrifield et al., J. Am. Chem. Soc., 1963, 85:2149</text></nplcit>, which involves assembling a linear peptide chain on a resin support using protected amino acids. Solid phase peptide synthesis typically utilizes either the Boc or Fmoc strategy, both of which are well known in the art.

0073The general strategy for the hybrid synthesis of SP-353 includes solid phase and solution phase syntheses to produce suitable peptide fragments (see Schemes 5 and 6 below), subsequent segment condensation to form the linear crude peptide (see Scheme 7 below), and natural oxidative folding to form the cyclized final product (see Scheme 7 below). The same strategy can also be used to produce other ST peptide analogs (such as SP-354, linaclotide, etc.) of similar amino acid sequences shown in Table II. <chemistry id="chem0007" num="0007"><img file="EP4424697A2_D0007.tif" /></chemistry><chemistry id="chem0008" num="0008"><img file="EP4424697A2_D0008.tif" /></chemistry><chemistry id="chem0009" num="0009"><img file="EP4424697A2_D0009.tif" /></chemistry>

0074The general strategy for the hybrid synthesis of SP-333 includes solid phase and solution phase syntheses to produce suitable peptide fragments (see Schemes 8 and 9 below), subsequent segment condensation to form the linear crude peptide (see Scheme 9 below), oxidative folding to form the cyclized final product, purification, and lyophilization (see Scheme 10 below). <chemistry id="chem0010" num="0010"><img file="EP4424697A2_D0010.tif" /></chemistry><chemistry id="chem0011" num="0011"><img file="EP4424697A2_D0011.tif" /></chemistry><chemistry id="chem0012" num="0012"><img file="EP4424697A2_D0012.tif" /></chemistry> understood that certain steps in Scheme 11 may be repeated (e.g., rinsing column with deionized water) or absent (e.g., salt exchange or alcohol removal after dewatering) to optimize the purification process. <chemistry id="chem0013" num="0013"><img file="EP4424697A2_D0013.tif" /></chemistry>

0075The precipitation process as illustrated in Scheme 11 provides a purified peptide, e.g., peptide GCC agonists. Preferably, the purified peptide is SP-304 (SEQ ID NO:1) or SP-333 (SEQ ID NO:9). In one embodiment, the purified SP-304 or SP-333 has a bulk density of no less than 0.05 g/mL, no less than 0.1 g/mL, no less than 0.2 g/mL, no less than 0.3 g/mL, no less than 0.4 g/mL, or no less than 0.5 g/mL. In a preferred embodiment, the 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 about 0.4-0.5 g/mL. In one embodiment, the purified SP-304 or SP-333 has a tap density of no less than 0.08 g/mL, no less than 0.1 g/mL, no less than 0.15 g/mL, no less than 0.2 g/mL, no less than 0.3 g/mL, no less than 0.4 g/mL, no less than 0.5 g/mL, or no less than 0.6 g/mL. For example, the purified SP-304 or SP-333 has a tap 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 peptide SP-304 or SP-333 contains &lt; 0.01% acetamide (e.g., &lt;28ppm), &lt;0.3% ammonium ion (e.g., &lt;0.25%), &lt;0.01% acetonitrile (e.g., &lt;20 ppm), and/or &lt; 0.1% TFA (e.g., ≤0.09%). In one embodiment, the purified peptide SP-304 or SP-333 has a bulk density of 0.4-0.5 g/mL, has a tap density of 0.6-0.7 g/mL, and contains &lt; 0.01% acetamide (e.g., &lt;28ppm), &lt;0.3% ammonium ion (e.g., &lt;0.25%), &lt;0.01% acetonitrile (e.g., &lt;20 ppm), and/or &lt; 0.1% TFA (e.g., ≤0.09%).

0076In some embodiments provides a purified peptide having the GCC agonist sequence selected from the group consisting of SEQ ID NOs: 1, 9, and 104, wherein the purified peptide has the following characteristics: <ol id="ol0002" compact="compact"><li>a) has a bulk density of not greater than 0.1 g/mL;</li><li>b) contains less than 50 ppm acetamide;</li><li>c) less than 0.3% alpha-Asp-9-plenacanatide.</li></ol>

0077The purified peptide can have one or more of the following features.

0078In one embodiment, the peptide is stable at 25⁰C for at least three months.

0079In one embodiment, the peptide has a particle size distribution having a D10 value of between about 2 tp 15 µm;a D50 value of between about 15-50 µm; and a D90 value of between about 40-80 µm when measured by light scattering with liquid dispersant.

0080In one embodiment, the purified peptide contains no more than 35 ppm acetamide (e.g., ≤ 18 ppm).

0081In one embodiment, the purified peptide contains less than 0.15% alpha-Asp-9-plenacanatide (which has a Relative Retention Time (RRT) of ~1.33 from the ultra-performance liquid chromatography (UPLC) analysis described herein).

0082In some embodiments, the purified peptide has a bulk density of not greater than 0.09 g/mL, not greater than 0.08 g/rnL, not greater than 0.07 g/mL, not greater than 0.06 g/mL, not greater than 0.05 g/mL, not greater than 0.04 g/mL, or not greater than 0.03 g/mL.

0083In some embodiments, the purified peptide is substantially free of water (e.g., water content not exceeding 10%, 9%, 8%,7%. 6%, 5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.5%, 0.25%, or 0.1%, of the total weight of the peptide).

0084In some embodiments, the purified peptide has a chromatographic purity of no less than 95%, no less than 96%, or no less than 97%.

0085In some embodiments,the total content of impurities in the purified peptide is less than 3% (e.g., &lt;2% or &lt;1%).

0086In some embodiments, the purified peptide is further substantially free of one ormore impurities selected from acetonitrile, alcohols, ammonium, acetates, and TFA.

0087For example, the purified peptide contains less than 300 ppm acetonitrile (e.g., &lt; 250 ppm).

0088In some embodiments, the purified peptide contains less than 0.2% TFA (e.g., &lt;0.15%, &lt;0.1%, &lt;400 ppm, &lt; 300 ppm, &lt; 200 ppm, &lt; 100 ppm, or &lt; 50 ppm).

0089In some embodiments, the purified peptide contains less than 0.2% isopropanol, i.e., IPA (e.g., &lt;0.15%, &lt; 0.1%, &lt; 1000 ppm, &lt; 900 ppm&lt; 800 ppm, &lt; 700 ppm, &lt; 600 ppm, &lt; 500 ppm, &lt; 400 ppm, &lt; 300 ppm, &lt; 200 ppm, &lt; 100 ppm, &lt; 50 ppm, or &lt; 20 ppm).

0090In some embodiments, the purified peptide contains less than 0.25 % acetate (e.g., &lt;0.2% or &lt;0.1%)).

0091In some embodiments, the purified peptide is substantially free of topoisomers (e.g., &lt;0.4%, &lt;0.3%, &lt;0.2% or &lt;0.1%).

0092In some embodiments, the purified peptide is substantially free of iso-Asp2-plecanatide (RRT 0.96- 0.97) (e.g., &lt;0.4%, &lt;0.3%, &lt;0.2% or &lt;0.1%).

0093In some embodiments,the purified peptide has a chromatographic purity of no less than 96%, no less than 97%, or no less than 98%. For example, the GCC agonist peptide has chromatographic impurity content of no greater than 4%, no greater than 3.5%, no greater than 3%, no greater than 2.5%, no greater than 2%, no greater than 1.5%, or no greater than 1%. The chromatographic impurity content is determined as total area percentages of impurities by HPLC. The chromatographic impurity content includes topoisomer content. The impurities do not include any pharmaceutically acceptable excipient used for drug formulation.

0094In some embodiments, the purified peptide is substantially free of contaminants resulted 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 contaminant content 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 weight of the peptide. For example, the purified peptide contains &lt; 50 ppm acetamide (e.g., &lt; 35 ppm or ≤ 18 ppm), &lt; 300 ppm acetonitrile (e.g., &lt; 250 ppm), &lt; 1000 ppm TFA (e.g., &lt; 400 ppm, &lt; 300 ppm, &lt; 200 ppm, &lt; 100 ppm, or &lt; 50 ppm), &lt;2000 ppm isopropanol (e.g., &lt;1500 ppm, &lt; 000 ppm, &lt;500 ppm, &lt; 400 ppm, &lt; 300 ppm, &lt; 200 ppm, &lt; 100 ppm, &lt; 50 ppm, or &lt; 20 ppm), , and/or &lt; 0.25 % acetate (e.g., &lt;0.2% or &lt;0.1%). The content of contaminants can be determined by conventional methods such as gas chromatography. Preferably, the residual solvents in the purified peptide of the invention are less than the limits set in the ICH guidelines, e.g., IMPURITIES: GUIDELINE FOR RESIDUAL SOLVENTS Q3C(R5) (available at http://vww.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q3C/Step4/Q 3C_R5_Step4.pdf).

0095In some embodiments, the purified peptide contains less than 0.3% (e.g., &lt; 0.15%) alpha-Asp-9-plenacanatide (RRT 1.33).

0096In some embodiments,the purified peptide has a bulk density of not greater than 0.09 g/mL, not greater than 0.08 g/mL, not greater than 0.07 g/mL, not greater than 0.06 g/mL, not greater than 0.05 g/mL, not greater than 0.04 g/mL, or not greater than 0.03 g/mL.

0097In some embodiments,the purified peptide is substantially free of iso-Asp2-plecanatide (RRT ~ 0.96-0.97). In this context "substantially" free of iso-Asp2-plecanatide means that the iso-Asp2-plecanatide 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 weight of the peptide.

0098In 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 weight of the peptide.

0099In 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%, of the total weight of the peptide.

0100For example, the purified peptide has a particle size distribution characterized by a D50 value of about 600 µm when measured by light scattering with air dispersant. In comparison, the peptides purified from the lyophilization and precipitation processes described in <patcit id="pcit0020" dnum="WO2012118972A"><text>WO2012/118972</text></patcit> have D50 values of about 180-250 µm and about 300 µm, respectively.

0101For example, the purified peptide prepared by the processes of the invention has a suitable size distribution for pharmaceutical formulation. In one embodiment, the peptide (e.g., SP-304) has a size distribution (e.g., an average size of 80-120 µm) comparable to that of the pharmaceutical excipient (e.g., microcrystalline cellulose) used in the formulation, for example, in the 3 mg/day unit dose form. The size distribution of the purified peptide may vary based on the unit dose. For example, when unit dose is lower than 3 mg/day, the purified peptide in the pharmaceutical formulation has a smaller average size than that in the 3 mg/day dose. For example, the purified peptide prepared by the processes of the invention is milled to reach the suitable size distribution.

0102The size distribution of the peptide of the invention can be determined by traditional methods, such as sieve analysis, light obscuration or dynamic light scattering analyses.

0103In one embodiment, the invention provides a purification process of purifying peptides, including those synthesized by the hybrid methods disclosed herein. The process includes, e.g., the steps (referred to as "isolation process 2") illustrated in Scheme 12 below, as compared to the isolation steps described in <patcit id="pcit0021" dnum="WO2012118972A"><text>WO2012/118972</text></patcit> (referred to as "isolation process 1"). <chemistry id="chem0014" num="0014"><img file="EP4424697A2_D0014.tif" /></chemistry>

0104In one embodiment, the purification process of the invention includes the following steps:

0105<b>Monocyclization of Linear Crude Peptide:</b> The disulfide bond between Cys⁴ and Cys¹² of SP-304 is first formed by H₂O₂ oxidation at 8.0-8.5 pH to form the monocyclized crude peptide. The linear crude peptide is slowly added and dissolving in 0.5% ammonium acetate in water buffer with pre-added H₂O₂ in a ratio of 100:9 gram of peptide to mL H₂O₂ to produce a final crude concentration of approximately 1.0mg/mL. The solution pH is then adjusted to 8.5 with an NH₄OH solution while stirring in the open air. The oxidative monocyclization reaction is monitored, e.g., using HPLC Method 2 as described in Example 8. When the area% of the linear crude peptide is ≤5.0% of the area% of monocyclized peptide, the oxidation reaction is stopped by adjusting the pH of peptide solution to 1.7-2 using a TFA solution. The peptide solution is then transferred to the next step for the formation of the dicyclized peptide.

0106<b>Dicyclization of Monocyclized Peptide:</b> The disulfide bond between the Cys7 and Cys15 is formed by 3.0% (w/v) I₂ in acetonitrile solution. The disulfide bridge is created while simultaneously removing the Acm side-chain protecting groups present on the remaining Cys residues. The oxidative dicyclization reaction is monitored, e.g., using HPLC Method 2. Excess iodine is quenched with a 0.1M ascorbic acid in water solution. Upon completion of the reaction, the dicyclized peptide is adjusted to pH 6.5-7 using an NH₄OH solution and the material is prepared for primary purification.

0107<b>Primary Purification of Dicyclized Crude Peptide:</b> The dicyclized crude peptide solution resulting from the oxidation steps is then loaded onto a preparative RP-HPLC column packed with C₁₈ reverse phase resin which is operated by a preparative HPLC system. The peptide is eluted in a 1% TEAP in Water, pH7/ACN buffer system. HPLC Method 1 as described in Example 8, for example, is used to ascertain the percent purity of fractions and pools obtained during the primary purification run.

0108<b>Recycle Pool(s) Purification of Dicyclized Peptide</b>: After primary purification, the fractions that require further purification are purified based on the purity of the traction pools using a 1%TFAP in Water, pH7/ACN or a 0.2% Acetic Acid in Water/ ACN buffer system. The dicyclized crude peptide solution resulting from the oxidation steps is then loaded onto a preparative RP-HPLC column packed with C₁₈ reverse phase resin which is operated by a preparative HPLC system. The peptide is eluted in a 1%TEAP in Water, pH7/ACN buffer system. HPLC Method 1 as described in Example 8, for example, is used to ascertain the percent purity of fractions and pools obtained during the Recycle Purification run.

0109<b>Secondary Purification and Salt Exchange:</b> After recycle purification, the fractions that require further purification are purified based on the purity of the fraction pools using a 1%TEAP in Water, pH7/ACN or a 0.2% Acetic Acid in Water/ ACN buffer system. HPLC Method 1 as described in Example 8, for example, is used to ascertain the percent purity of fractions and pools obtained during the Secondary Purification run. UPLC Method 1 as described in Example 8, for example, is used to ascertain the percent purity of Main Pool obtained during all the Purification run before move to next step.

0110<b>Solvent =Exchange:</b> Material meeting main pool criteria is loaded onto the preparative RP-HPLC column at the flange end in the reverse direction, washed with a water/isopropanol solution (e.g., with a water to isopropanol ratio of 99: 1) from the reverse direction, and eluted with a water/isopropanol solution (e.g., with a water to isopropanol ratio of 60:40) in the forward direction. The collected peptide solution is tested by UPLC Method 1 to ascertain the purity, then the peptide solution is filtered, undergoes rotary evaporation to remove excess isopropanol (to e.g., below 5%), followed by sublot lyophilization for, e.g., no less than 96 hours.

0111<b>Reconstitution and Final Lyophilization:</b> The sublot lyophilized dry peptide undergoes reconstitution in water to form a homogenous lot. A buffer, such as ammonium acetate (e.g., 0.5% (w/w) to dry peptide), is added to the solution and it is mixed until the ammonium acetate and the peptide are dissolved. The material can be analyzed by UPLC Method 1 to verify the purity. The dissolved material can be installed onto the tray lyophilizer and kept under vacuum for, e.g., no less than 120 hours to comprise the final dry peptide material.

1.1 GCC

Agonists

0112The GCC agonists prepared by the processes of the invention can bind to guanylate cyclase C and stimulate intracellular production of cGMP. Optionally, the GCC agonists induce apoptosis and inhibit proliferation of epithelial cells. The term, "guanylate cyclase C" refers to a transmembrane form of guanylate cyclase that acts as the intestinal receptor for the heat-stable toxin (ST) peptides secreted by enteric bacteria. Guanylate cyclase C is also the receptor for the naturally occurring peptides guanylin and uroguanylin. The possibility that there may be different receptors for each of these peptides has not been excluded. Hence, the term "guanylate cyclase C" may also encompass a class of transmembrane guanylate cyclase receptors expressed on epithelial cells lining the gastrointestinal mucosa.

0113The term "GCC agonist" refers to both peptides and non-peptide compounds such as that bind to an intestinal guanylate cyclase C and stimulate the intracellular production of cGMP. Where the GCC agonist is a peptide, the term encompasses biologically active fragments of such peptides and pro-peptides that bind to guanylate cyclase C and stimulate the intracellular production of cGMP.

1.1.1 GCC Agonist Peptides

0114The GCC agonists suitable for the methods of the invention are preferably peptides. In some embodiments, the GCC agonist peptide is less than 30 amino acids in length. In particular embodiments, the GCC agonist peptide is less than or equal to 30, 25, 20, 15, 14, 13, 12, 11, 10, or 5 amino acids in length. Examples of GCC agonist peptides for use in the formulations and methods of the invention include those described in <patcit id="pcit0022" dnum="US7879802B"><text>U.S. Patent Nos. 7,879,802</text></patcit> and <patcit id="pcit0023" dnum="US8034782B"><text>8,034,782</text></patcit>, and U.S. Publication Nos. <patcit id="pcit0024" dnum="US20100069306A"><text>US 2010-0069306</text></patcit> and <patcit id="pcit0025" dnum="US20100120694A"><text>US 2010-0120694</text></patcit>, each of which is incorporated by reference herein in its entirety.

0115Specific 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 Tables I-VII, the terms "PEG3" or "3PEG" refer to a polyethylene glycol such as aminoethyloxy-ethyloxy-acetic acid (AeeA), and polymers thereof. As used herein, the term "AMIDE" is meant to denote 5 that the terminal carboxylic acid is replaced with an amide group, i.e., the terminal COOH is replaced with CONH₂.

0116The term "X_aa" refers to any natural or unnatural amino acid or amino acid analogue. The term "M_aa" refers to a cysteine (Cys), penicillamine (Pen) homocysteine, or 3-mercaptoproline. The term "Xaa_n1" is meant to denote an amino acid sequence of any natural or unnatural amino acid or amino acid analogue that is one, two or three residues in length; Xaa_n2 is meant to denote an amino acid sequence that is zero or one residue in length; and Xaa_n3 is meant to denote an amino acid sequence zero, one, two, three, four , five or six residues in length. Additionally, any amino acid represented by Xaa, Xaa_n1, Xaa_n2, or Xaa_n3 may be an L-amino acid, a D-amino acid, a methylated amino acid or any combination of thereof. Optionally, any GCC agonist peptide represented by Formulas I to XX in the tables may contain one or more polyethylene glycol residues at the N- terminus, C-terminus or both.

0117In certain embodiments, a GCC agonist purified or prepared by the methods of the invention comprises a peptide selected from SEQ ID NOs: 1-251, the sequences of which are set forth below in Tables I to VII below. In one embodiment, a GCC agonist purified by the methods of the invention comprises the peptide designated by SEQ ID NOs: 1, 9, or 104.

0118In certain embodiments, a GCC agonist prepared by the methods of the invention comprises a peptide that is substantially equivalent to a peptide selected from SEQ ID NOs: 1-251. The term "substantially equivalent" refers to a peptide that has an amino acid sequence equivalent to that of the binding domain where certain residues may be deleted or replaced with other amino acids without impairing the peptide's ability to bind to an intestinal guanylate cyclase receptor and stimulate fluid and electrolyte transport.

0119In certain embodimenst, a GCC agonist prepared by the methods of the invention comprises a peptide that are analogs to a peptide selected from SEQ ID NOs: 1-251. Particularly, these analogs contain an α-aminoadipic acid (Aad), preferably at the 3^rd position from the N-terminus of each peptide or at the position to the N-terminal side next to the first cysteine ("Cys") residue.251

0120In certain embodiments, the GCC agonist peptides are analogues of uroguanylin or a bacterial ST peptide. Uroguanylin is a circulating peptide hormone with natriuretic activity. An ST peptide is a member of a family of heat stable enterotoxins (ST peptides) secreted by pathogenic strains of <i>E. coli</i> and other enteric bacteria that activate guanylate cyclase receptor and cause secretory diarrhea. Unlike bacterial ST peptides, the binding of uroguanylin to guanylate cyclase receptor is dependent on the physiological pH of the gut. Therefore, uroguanylin is expected to regulate fluid and electrolyte transport in a pH dependent manner and without causing severe diarrhea.

0121The GCC agonist peptides prepared by the methods of the invention can 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-inverso 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. <i>See, e.g.,</i><nplcit id="ncit0012" npl-type="s"><text>Jameson et al., Nature, 368, 744-746 (1994</text></nplcit>); <nplcit id="ncit0013" npl-type="s"><text>Brady et al., Nature, 368, 692-693 (1994</text></nplcit>). The net result of conibining D-enantiomers and reverse synthesis is that the positions of carbonyl and amino groups in each amide bond are exchanged, while the position of the side-chain groups at each alpha carbon is preserved. Unless specifically stated otherwise, it is presumed that any given L-amino acid sequence of the invention may be made into a D retro-inverso peptide by synthesizing a reverse of the sequence for the corresponding native L-amino acid sequence.

0122The GCC agonist peptides prepared by the methods of the invention are able to induce 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 naturally occurring GCC agonists such as uroguanylin, guanylin, or ST peptides. Optionally, the GCC agonist peptide stimulates 5%, 10%, 20%, 30%, 40%, 50% , 75%, 90% or more intracellular cGMP compared to SP-304 (SEQ ID NO:1). In further embodiments, the GCC agonist peptide stimulates apoptosis, e.g., programmed cell death, or activate the cystic fibrosis transmembrane conductance regulator (CFTR).

0123In some embodiments, the GCC agonist peptides prepared by the methods of the invention are more stable than naturally occurring GCC agonists and/or SP-304 (SEQ ID NO:1), SP-339 (linaclotide) (SEQ ID NO: 55) or SP-340 (SEQ ID NO: 56). For example, the GCC agonist peptide degrades 2%, 3%, 5%, 10%, 15%, 20%, 30%, 40%, 50% , 75%, 90% or less compared to naturally occurring GCC agonists and/or SP-304, SP-339 (linaclotide) or SP-340. In certain embodiments, the GCC agonist peptides for use in the formulations and methods of the invention are more stable to proteolytic digestion than naturally occurring 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, a GCC agonist peptide is pegylated in order to render the peptides more resistant towards proteolysis by enzymes of the gastrointestinal tract. In a preferred embodiment, the GCC agonist peptide is pegylated with the aminoethyloxy-ethyloxy-acetic acid (Aeea) group at its C-terminal end, at its N-terminal end, or at both termini.

0124Specific examples of GCC agonist peptides that can be prepared by the methods of the invention include a peptide selected from the group designated by SEQ ID NOs: 1-251.

0125In one embodiment, the GCC agonist peptide is a peptide having the amino acid sequence of any one of Formulas X- XVII (e.g. SEQ ID NOs: 87-98).

0126In some embodiments, GCC agonist peptides include peptides having the amino acid sequence of Formula I, wherein at least one amino acid of Formula I is a D-amino acid or a methylated amino acid and/or the amino acid at position 16 is a serine. 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 a d-leucine or a 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, Asn¹, Asp² or Glu³ (or a combination thereof) of Formula I is a D-amino acid or a methylated amino acid. Preferably, the amino acid at position Xaa⁶ of Formula I is a leucine, serine or tyrosine.

0127In alternative embodiments, GCC agonist peptides include peptides having the 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 denoted by Xaa_n2 of Formula II is a D-amino acid or a methylated amino acid. In some embodiments, the amino acid denoted by Xaa_n2 of Formula II is a leucine, a d-leucine, a serine, or a d-serine. Preferably, the one or more amino acids denoted by Xaa_n1 of Formula II is a D-amino acid or a methylated amino acid. Preferably, the amino acid at position Xaa⁶ of Formula II is a leucine, a serine, or a tyrosine.

0128In some embodiments, GCC agonist peptides include peptides having the amino acid sequence of Formula III, wherein at least one amino acid of Formula III is a D-amino acid or a methylated amino acid and/or Maa is not a cysteine. Preferably, the amino acid denoted by Xaa_n2 of Formula III is a D-amino acid or a methylated amino acid. In some embodiments the amino acid denoted by Xaa_n2 of Formula III is a leucine, a d-leucine, a serine, or a d-serine. Preferably, the one or more amino acids denoted by Xaa_n1 of Formula III is a D-amino acid or a methylated amino acid. Preferably, the amino acid at position Xaa⁶ of FFormula III is a leucine, a serine, or a tyrosine.

0129In other embodiments, GCC agonist peptides include peptides having the amino acid sequence of Formula IV, wherein at least one amino acid of Formula IV is a D-amino acid or a methylated amino acid, and/or Maa is not a cysteine. Preferably, the Xaa_n2 of Formula IV is a D-amino acid or a methylated amino acid. In some embodiments, the amino acid denoted by Xaa_n2 of Formula IV is a leucine, a d-leucine, a serine, or a d-serine. Preferably, the one or more of the amino acids denoted by Xaa_n1 of Formula IV is a D-amino acid or a methylated amino acid. Preferably, the amino acid denoted Xaa⁶ of Formula IV is a leucine, a serine, or a tyrosine.

0130In further embodiments, GCC agonist peptides include peptides having the amino acid sequence of Formula V, wherein at least one amino acid of Formula V 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 (i.e., Xaa¹⁶) of Formula V is a d-leucine or a d-serine. Optionally, one or more of the amino acids at position 1-3 of Formula V are D-amino acids or methylated amino acids or a combination of D-amino acids or methylated amino acids. For example, Asn¹, Asp² or Glu³ (or a combination thereof) of Formula V is a D-amino acids or a methylated amino acid. Preferably, the amino acid denoted at Xaa⁶ of Formula V is a leucine, a serine, or a tyrosine.

0131In additional embodiments, 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 a leucine, a serine, or a tyrosine. In some aspects the amino acid at position 16 of Formula VI, VII, VIII, or IX is a leucine or a serine. Preferably, the amino acid at position 16 of Formula V is a D-amino acid or a methylated amino acid.

0132In additional embodiments, 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 is a D-amino acid or a methylated amino acid. Preferably, the amino acid at the carboxy terminus of the peptides according to 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 according to Formulas X, XI, XII, XIII, XIV, XV, XVI or XVII is a D-tyrosine.

0133Preferably, the amino acid denoted by Xaa⁶ of Formula XIV is a tyrosine, phenylalanine or a serine. Most preferably the amino acid denoted by Xaa⁶ of Formula XIV is a phenylalanine or a serine. Preferably, the amino acid denoted by Xaa⁴ of Formula XV, XVI or XVII is a tyrosine, a phenylalanine, or a serine. Most preferably, the amino acid position Xaa⁴ of Formula V, XVI or XVII is a phenylalanine or a serine.

0134In some embodiments, GCRA peptides include peptides containing the amino acid sequence of Formula XVIII. Preferably, the amino acid at position 1 of Formula XVIII is a glutamic acid, aspartic acid, glutamine or lysine. Preferably, the amino acid at position 2 and 3 of Formula XVIII is a glutamic acid, or an aspartic acid. Preferably, the amino acid at position 5 a glutamic acid. Preferably, the amino acid at position 6 of Formula XVIII is an isoleucine, valine, serine, threonine, or tyrosine. Preferably, the amino acid at position 8 of Formula XVIII is a valine or isoleucine. Preferably, the amino acid at position 9 of Formula XVIII is an asparagine. Preferably, the amino acid at position 10 of Formula XVIII is a valine or a methionine. Preferably, the amino acid at position 11 of Formula XVIII is an alanine. Preferably, the amino acid at position 13 of Formula XVIII I is a threonine. Preferably, the amino acid at position 14 of Formula XVIII is a glycine. Preferably, the amino acid at position 16 of Formula XVIII is a leucine, serine or threonine

0135In alternative embodiments, GCRA peptides include peptides containing the amino acid sequence of Formula XIX. Preferably, the amino acid at position 1 of Formula XIX is a serine or asparagine. Preferably, the amino acid at position 2 of Formula XIX is a histidine or an aspartic acid. Preferably, the amino acid at position 3 of Formula XIX is a threonine or a glutamic acid. Preferably, the amino acid at position 5 of Formula XIX is a glutamic acid. Preferably, the amino acid at position 6 of Formula XIX is an isoleucine, leucine, valine, or tyrosine. Preferably, the amino acid at position 8, 10, 11, or 13 of Formula XIX is an alanine. Preferably, the amino acid at position 9 of Formula XIX is an asparagine or a phenylalanine. Preferably, the amino acid at position 14 of Formula XIX is a glycine.

0136In further embodiments, GCRA peptides include peptides containing the amino acid sequence of Formula XX. Preferably, the amino acid at position 1 of Formula XX is a glutamine. Preferably, the amino acid at position 2 or 3 of Formula XX is a glutamic acid or an aspartic acid. Preferably, the amino acid at position 5 of Formula XX is a 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 a threonione. Preferably, the amino acid at position 1 of Formula XX is a glycine. Preferably, the amino acid at position 15 of Formula XX is a tyrosine. Optionally, the amino acid at position 15 of Formula XX is two amino acid in length and is Cysteine (Cys), Penicillamine (Pen) homocysteine, or 3-mercaptoproline and serine, leucine or threonine.

0137In certain embodiments, one or more amino acids of the GCC agonist peptides are replaced by a non-naturally occurring amino acid or a naturally or non-naturally occurring amino acid analog. Such amino acids and amino acid analogs are known in the art. See, for example, <nplcit id="ncit0014" npl-type="b"><text>Hunt, "The Non-Protein Amino Acids," in Chemistry and Biochemistry of the Amino Acids, Barrett, Chapman, and Hall, 1985</text></nplcit>. In some embodiments, an amino acid is replaced by a naturally-occurring, non-essential amino acid, e.g., taurine. Non-limiting examples of naturally occurring amino acids that can be replaced by non-protein amino acids include the following: (1) an aromatic amino acid can be replaced by 3,4-dihydroxy-L-phenylalanine, 3-iodo-L-tyrosine, triiodothyronine, L-thyroxine, phenylglycine (Phg) or nor-tyrosine (norTyr); (2) Phg and norTyr and other amino acids including Plue and Tyr can be substituted by, <i>e.g.,</i> a halogen, -CH3, -OH, - CH2NH3, -C(O)H, -CH2CH3, - CN, -CH2CH2CH3, -SH, or another group; (3) glutamine residues can be substituted with gamma-Hydroxy-Glu or gamma- Carboxy-Glu; (4) tyrosine residues can be substituted with an alpha substituted amino acid such as L-alpha-methylphenylalanine or by analogues such as: 3-Amino-Tyr; Tyr(CH3); Tyr(PO3(CH3)2); Tyr(SO3H); beta-Cyclohexyl-Ala; beta-(1-Cyclopentenyl)-Ala; beta- Cyclopentyl-Ala; beta-Cyclopropyl-Ala; beta-Quinolyl-Ala; beta-(2-Thiazolyl)-Ala; beta- (Triazole-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 substituted with homopro (L-pipecolic acid); hydroxy-Pro; 3,4-Dehydro-Pro; 4-fluoro-Pro; or alpha-methyl-Pro or an N(alpha)-C(alpha) cyclized amino acid analogues with the structure: n = 0, 1, 2, 3; and (6) alanine residues can be substituted with alpha-substituted or N-methylated amino acid such as alpha-amino isobutyric 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. Alanine can also be substituted with: n = 0, 1, 2, 3 Glycine residues can be substituted with alpha-amino isobutyric acid (aib) or L/D-alpha- ethylalanine (L/D-isovaline).

0138Further examples of non-natural amino acids include: an unnatural analog of tyrosine; an unnatural analogue of glutamine; an unnatural analogue of phenylalanine; an unnatural analogue of serine; an unnatural analogue of threonine; an alkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynyl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino substituted amino acid, or any combination thereof; an amino acid with a photoactivatable cross-linker; a spin-labeled amino acid; a fluorescent amino acid; an amino acid with a novel functional group; an amino acid that covalently or noncovalently interacts with another molecule; a metal binding amino acid; an amino acid that is amidated at a site that is not naturally amidated, a metal-containing amino acid; a radioactive amino acid; a photocaged and/or photoisomerizable amino acid; a biotin or biotin-analogue containing amino acid; a glycosylated or carbohydrate modified amino acid; a keto containing amino acid; amino acids comprising polyethylene glycol or polyether; a heavy atom substituted amino acid (e.g., an amino acid containing deuterium, tritium, ¹³C, ¹⁵N, or ¹⁸O); a chemically cleavable or photocleavable amino acid; an amino acid with an elongated side chain; an amino acid containing a toxic group; a sugar substituted amino acid, <i>e.g.,</i> a sugar substituted serine or the like; a carbon-linked sugar-containing amino acid; a redox-active amino acid; an α-hydroxy containing acid; an amino thio acid containing amino acid; an α, α disubstituted amino acid; a β-amino acid; a cyclic amino acid other than proline; an O-methyl-L-tyrosine; an L-3-(2-naphthyl)alanine; a 3-methyl-phenylalanine; a ρ-acetyl-L-phenylalanine; an O-4-allyl-L-tyrosine; a 4-propyl-L-tyrosine; a tri-O-acetyl-GlcNAc β -serine; an L-Dopa; a fluorinated phenylalanine; an isopropyl-L-phenylalanine; a p-azido-L-phenylalanine; a p-acyl-L-phenylalanine; a p-benzoyl-L-phenylalanine; an L-phosphoserine; a phosphonoserine; a phosphonotyrosine; a p-iodo-phenylalanine; a 4-fluorophenylglycine; a p-bromophenylalanine; a p-amino-L-phenylalanine; an isopropyl-L-phenylalanine; L-3-(2-naphthyl)alanine; D- 3-(2-naphthyl)alanine (dNal); an amino-, isopropyl-, or O-allyl-containing phenylalanine analogue; a dopa, 0-methyl-L-tyrosine; a glycosylated amino acid; a p-(propargyloxy)phenylalanine; dimethyl-Lysine; hydroxy-proline; mercaptopropionic acid; methyl-lysine; 3-nitro-tyrosine; norleucine; pyro-glutamic acid; Z (Carbobenzoxyl); ε-Acetyl-Lysine; β-alanine; aminobenzoyl derivative; aminobutyric acid (Abu); citrulline; aminohexanoic acid; aminoisobutyric acid (AIB); cyclohexylalanine; d-cyclohexylalanine; hydroxyproline; nitro-arginine; nitro-phenylalanine; nitro-tyrosine; norvaline; octahydroindole carboxylate; ornithine (Orn); penicillamine (PEN); tetrahydroisoquinoline; acetamidomethyl protected amino acids and pegylated amino acids. Further examples of unnatural amino acids and amino acid analogs can be found in <patcit id="pcit0026" dnum="US20030108885A"><text>U.S. 20030108885</text></patcit>, <patcit id="pcit0027" dnum="US20030082575A"><text>U.S. 20030082575</text></patcit>, <patcit id="pcit0028" dnum="US20060019347A"><text>US20060019347</text></patcit> (paragraphs 410-418) and the references cited therein. The polypeptides of the invention can include further modifications including those described in <patcit id="pcit0029" dnum="US20060019347A"><text>US20060019347</text></patcit>, paragraph 589. Exemplary GCC agonist peptides which include a non-naturally occurring amino acid include for example SP-368 and SP-369.

0139In some embodiments, the GCC agonist peptides are cyclic peptides. GCC agonist cyclic peptides can be prepared by methods known in the art. For example, macrocyclization is often accomplished by forming an amide bond between the peptide N- and C-termini, between a side chain and the N- or C-terminus [e.g., with K₃Fe(CN)₆ at pH 8.5] (<nplcit id="ncit0015" npl-type="s"><text>Samson et al., Endocrinology, 137: 5182-5185 (1996</text></nplcit>)), or between two amino acid side chains, such as cysteine. See, <i>e.g.,</i><nplcit id="ncit0016" npl-type="s"><text>DeGrado, Adv Protein Chem, 39: 51-124 (1988</text></nplcit>). In various embodiments, the GCC agonist peptides are [4,12; 7,15] bicycles.

0140In certain embodiments, one or both Cys residues which normally form a disulfide bond in a GCC agonist peptide are replaced with homocysteine, penicillamine, 3-mercaptoproline (<nplcit id="ncit0017" npl-type="s"><text>Kolodziej et al. 1996 Int. J. Pept. Protein Res. 48:274</text></nplcit>), β, β dimethylcysteine (<nplcit id="ncit0018" npl-type="s"><text>Hunt et al. 1993 Int. J. Pept. Protein Res. 42:249</text></nplcit>), or diaminopropionic acid (<nplcit id="ncit0019" npl-type="s"><text>Smith et al. 1978 J. Med, Chem. 2 1:117</text></nplcit>) to form alternative internal cross-links at the positions of the normal disulfide bonds.

0141In certain embodiments, one or more disulfide bonds in a GCC agonist peptide are replaced by alternative covalent cross-links, e.g., an amide linkage (-CH₂CH(O)NHCH₂- or - CH₂NHCH(O)CH₂-), an ester linkage, a thioester linkage, a lactam bridge, a carbamoyl linkage, a urea linkage, a thiourea linkage, a phosphonate ester linkage, an alkyl linkage (-CH₂CH₂CH₂CH₂-), an alkenyl linkage (-CH₂CH=CHCH₂-), an ether linkage (-CH₂CH₂OCH₂- or -CH₂OCH₂CH₂-), a thioether linkage (-CH₂CH₂SCH₂- or - CH₂SCH₂CH₂-), an amine linkage (-CH₂CH₂NHCH₂- or -CH₂NHCH₂CH₂-) or a thioamide linkage (-CH₂C(S)NHCH₂- or -CH₂NHC (S)CH₂-). For example, <nplcit id="ncit0020" npl-type="s"><text>Ledu et al. (Proc. Natl. Acad. Sci. 100:11263-78, 2003</text></nplcit>) describe methods for preparing lactam and amide cross-links. Exemplary GCC agonist peptides which include a lactam bridge include, for example, SP-370.

0142In certain embodiments, the GCC agonist peptides have one or more conventional polypeptide bonds replaced by an alternative bond. Such replacements can increase the stability of the polypeptide. For example, replacement of the polypeptide bond between a residue amino terminal to an aromatic residue (<i>e.g.</i> Tyr, Phe, Trp) with an alternative bond can reduce cleavage by carboxy peptidases and may increase half-life in the digestive tract. Bonds that can replace polypeptide bonds include: a retro-inverso bond (C(O)-NH instead of NH-C(O); a reduced amide bond (NH-CH₂); a 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-olefine bond (CH=CH); a fluoro substituted trans-olefine bond (CF=CH); a ketomethylene bond (C(O)-CHR or CHR-C(O) wherein R is H or CH₃; and a fluoro-ketomethylene bond (C(O)-CFR or CFR-C(O) wherein R is H or F or CH₃.

0143In certain embodiments, the GCC agonist peptides are modified using standard modifications. Modifications may occur at the amino (N-), carboxy (C-) terminus, internally or a combination of any of the preceding. In one aspect described herein, there may be more than one type of modification on the polypeptide. Modifications include but are not limited to: acetylation, amidation, biotinylation, cinnamoylation, famesylation, formylation, myristoylation, palmitoylation, phosphorylation (Ser, Tyr or Thr), stearoylation, succinylation, sulfurylation and cyclisation (via disulfide bridges or amide cyclisation), and modification by Cys3 or Cys5. The GCC agonist peptides described herein may also be modified by 2, 4-dinitrophenyl (DNP), DNP-lysine, modification by 7-Amino-4-methyl- coumarin (AMC), flourescein, NBD (7-Nitrobenz-2-Oxa-1,3-Diazole), p-nitro-anilide, 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 may also be conjugated to, for example, polyethylene glycol (PEG); alkyl groups (e.g., C1-C20 straight or branched alkyl groups); fatty acid moieties; combinations of PEG, alkyl groups and fatty acid moieties (<i>See,</i><patcit id="pcit0030" dnum="US6309633B"><text>U.S. Patent 6,309,633</text></patcit>; <nplcit id="ncit0021" npl-type="s"><text>Soltero et al., 2001 Innovations in Pharmaceutical Technology 106-110</text></nplcit>); BSA and KLH (Keyhole Limpet Hemocyanin). The addition of PEG and other polymers which can be used to modify polypeptides of the invention is described in <patcit id="pcit0031" dnum="US2006019347A"><text>US20060 19347</text></patcit> section IX.

0144A GCC agonist peptide can also be a derivative of a GCC agonist peptide described herein. For example, a derivative includes hybrid and modified forms of GCC agonist peptides in which certain amino acids have been deleted or replaced. A modification may also include glycosylation. Preferrably, where the modification is an amino acid substitution, it is a conservative substitution at one or more positions that are predicted to be non-essential amino acid residues for the biological activity of the peptide. A "conservative substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (<i>e.g</i>., lysine, arginine, histidine), acidic side chains (<i>e.g.,</i> aspartic acid, glutamic acid), uncharged polar side chains (<i>e.g.,</i> glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (<i>e.g.,</i> alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (<i>e.g.,</i> threonine, valine, isoleucine) and aromatic side chains (<i>e.g</i>., tyrosine, phenylalanine, tryptophan, histidine).

0145In one embodiment, a GCC agonist peptide prepared by the methods described herein is subjected to random mutagenesis in order to identify mutants having biological activity.

0146In one embodiment, the methods of the invention can be used to prepare a GCC agonist peptide that is substantially homologous to a GCC agonist peptide described herein. Such substantially homologous peptides can be isolated by virtue of cross-reactivity with antibodies to a GCC agonist peptide described herein.

0147Further examples of GCC agonist peptides that can be prepared by the methods of the invention are found in Tables I-VII below.

1.1.2 Alternative Preparation Methods of GCC agonist peptides and Their Fragments

0148GCC agonist peptides and their fragments can be prepared using art recognized techniques such as molecular cloning, peptide synthesis, or site-directed mutagenesis.

0149In addition to the conventional solution- or solid-phase peptide synthesis described above, the GCC agonist peptides or their fragments can be produced by modem cloning techniques. For example, the GCC agonist peptides are produced either in bacteria including, without limitation, E. coli, or in other existing systems for polypeptide or protein production (e.g., Bacillus subtilis, baculovirus expression systems using Drosophila Sf9 cells, yeast or filamentous fungal expression systems, mammalian cell expression systems), or they can be chemically synthesized. If the GCC agonist peptide or variant peptide is to be produced in bacteria, e.g., E. coli, the nucleic acid molecule encoding the polypeptide may also encode a leader sequence that permits the secretion of the mature polypeptide from the cell. Thus, the sequence encoding the polypeptide can include the pre sequence and the pro sequence of, for example, a naturally-occurring bacterial ST polypeptide. The secreted, mature polypeptide can be purified from the culture medium.

0150The sequence encoding a GCC agonist peptide described herein can be inserted into a vector capable of delivering and maintaining the nucleic acid molecule in a bacterial cell. The DNA molecule may be inserted into an autonomously replicating vector (suitable vectors include, for example, pGEM3Z and pcDNA3, and derivatives thereof). The vector nucleic acid may be a bacterial or bacteriophage DNA such as bacteriophage lambda or M13 and derivatives thereof. Construction of a vector containing a nucleic acid described herein can 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 encoding nucleic acid molecule, elements that allow expression, such as a promoter and regulatory sequences. The expression vectors may contain transcriptional control sequences that control transcriptional initiation, such as promoter, enhancer, operator, and repressor sequences.

0151A variety of transcriptional control sequences are well known to those in the art. The expression vector can also include a translation regulatory sequence (e.g., an untranslated 5' sequence, an untranslated 3' sequence, or an internal ribosome entry site). The vector can be capable of autonomous replication or it can integrate into host DNA to ensure stability during polypeptide production.

0152The protein coding sequence that includes a 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 E binding protein, protein A, FLAG tag, hexa-histidine, myc tag or the influenza HA tag, in order to facilitate purification. The affinity tag or reporter fusion joins 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 generated. Expression of the fusion gene results in translation of a single polypeptide that includes both the polypeptide of interest and the affinity tag. In some instances where affinity tags are utilized, DNA sequence encoding a protease recognition site will be fused between the reading frames for the affinity tag and the polypeptide of interest.

0153Genetic constructs and methods suitable for production of immature and mature forms of the GCC agonist peptides and variants described herein in protein expression systems other than bacteria, and well known to those skilled in the art, can also be used to produce polypeptides in a biological system.

0154The peptides disclosed herein may be modified by attachment of a second molecule that confers a desired property upon the peptide, such as increased half-life in the body, for example, pegylation. Such modifications also fall within the scope of the term "variant" as used herein. <tables id="tabl0001" num="0001"><img file="EP4424697A2_D0015.tif" /></tables><tables id="tabl0002" num="0002"><img file="EP4424697A2_D0016.tif" /></tables><tables id="tabl0003" num="0003"><img file="EP4424697A2_D0017.tif" /></tables><tables id="tabl0004" num="0004"><img file="EP4424697A2_D0018.tif" /></tables><tables id="tabl0005" num="0005"><img file="EP4424697A2_D0019.tif" /></tables><tables id="tabl0006" num="0006"><img file="EP4424697A2_D0020.tif" /></tables><tables id="tabl0007" num="0007"><img file="EP4424697A2_D0021.tif" /></tables><tables id="tabl0008" num="0008"><img file="EP4424697A2_D0022.tif" /></tables><tables id="tabl0009" num="0009"><img file="EP4424697A2_D0023.tif" /></tables><tables id="tabl0010" num="0010"><img file="EP4424697A2_D0024.tif" /></tables><tables id="tabl0011" num="0011"><img file="EP4424697A2_D0025.tif" /></tables><tables id="tabl0012" num="0012"><img file="EP4424697A2_D0026.tif" /></tables><tables id="tabl0013" num="0013"><img file="EP4424697A2_D0027.tif" /></tables><tables id="tabl0014" num="0014"><img file="EP4424697A2_D0028.tif" /></tables><tables id="tabl0015" num="0015"><img file="EP4424697A2_D0029.tif" /></tables><tables id="tabl0016" num="0016"><img file="EP4424697A2_D0030.tif" /></tables><tables id="tabl0017" num="0017"><img file="EP4424697A2_D0031.tif" /></tables><tables id="tabl0018" num="0018"><img file="EP4424697A2_D0032.tif" /></tables><tables id="tabl0019" num="0019"><img file="EP4424697A2_D0033.tif" /></tables><tables id="tabl0020" num="0020"><img file="EP4424697A2_D0034.tif" /></tables><tables id="tabl0021" num="0021"><img file="EP4424697A2_D0035.tif" /></tables><tables id="tabl0022" num="0022"><img file="EP4424697A2_D0036.tif" /></tables>

1.2 Methods of Use

0155The invention provides 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 a formulation thereof to the subject. Non-limiting examples of gastrointestinal disorders that can be treated or prevented according to the methods of the invention include irritable bowel syndrome (IBS), non-ulcer dyspepsia, 1-1. pylori infection related ulcers, chronic intestinal pseudo-obstruction, functional dyspepsia, colonic pseudo-obstruction, duodenogastric reflux, gastroesophageal reflux disease (GERD), ileus (<i>e.g.,</i> postoperative ileus), gastroparesis, heartburn (high acidity in the GI tract), constipation (e.g., constipation associated with use of medications such as opioids, osteoarthritis drugs, or osteoporosis drugs); post surgical constipation, constipation associated with neuropathic disorders, Crohn's disease, and ulcerative colitis.

0156In one embodiment, the invention provides methods for treating or preventing gastrointestinal motility disorder, irritable bowel syndrome, a functional gastrointestinal disorder, gastroesophageal reflux disease, duodenogastric reflux, functional heartburn, dyspepsia, functional dyspepsia, non-ulcer dyspepsia, gastroparesis, chronic intestinal pseudo-obstruction, colonic pseudo-obstruction, obesity, congestive heart failure, or benign prostatic hyperplasia.

0157In one embodiment, the invention provides 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 a formulation thereof to the subject. Clinically accepted criteria that define constipation range from the frequency of bowel movements, the consistency of feces and the ease of bowel movement. One common definition of constipation is less than three bowel movements per week. Other definitions include abnormally hard stools or defecation that requires excessive straining (<nplcit id="ncit0022" npl-type="s"><text>Schiller 2001 Aliment Pharmacol Ther 15:749-763</text></nplcit>). Constipation may be idiopathic (functional constipation or slow transit constipation) or secondary to other causes including neurologic, metabolic or endocrine disorders. These disorders include diabetes mellitus, hypothyroidism, hyperthyroidism, hypocalcaemia, Multiple sclerosis, Parkinson's disease, spinal cord lesions, Neurofibromatosis, autonomic neuropathy, Chagas disease, Hirschsprung disease and cystic fibrosis. Constipation may also be the result of surgery or due to the use of drugs such as analgesics (like opioids), antihypertensives, anticonvulsants, antidepressants, antispasmodics and antipsychotics.

0158In various embodiments, the constipation is associated with use of a therapeutic agent; the constipation is associated with a neuropathic disorder; the constipation is postsurgical constipation; the constipation is associated with a gastrointestinal disorder; the constipation is idiopathic (functional constipation or slow transit constipation); the constipation is associated with neuropathic, metabolic or endocrine disorder (e.g., diabetes mellitus, hypothyroidism, hyperthyroidism, hypocalcaemia, Multiple Sclerosis, Parkinson's disease, spinal cord lesions, neurofibromatosis, autonomic neuropathy, Chagas disease, Hirschsprung disease or cystic fibrosis). Constipation may also be the result of surgery or due to the use of drugs such as analgesics (e.g., opioids), antihypertensives, anticonvulsants, antidepressants, antispasmodics and antipsychotics.

0159In one embodiment, the invention provides 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 a formulation thereof to the subject.

0160The term "treating" as used herein refers to a reduction, a partial improvement, amelioration, or a mitigation of at least one clinical symptom associated with the gastrointestinal disorders being treated. The term "preventing" refers to an inhibition or delay in the onset or progression of at least one clinical symptom associated with the gastrointestinal disorders to be prevented. The term "effective amount" as used herein refers to an amount that provides some improvement or benefit to the subject. In certain embodiments, an effective amount is an amount that provides some alleviation, mitigation, and/or decrease in at least one clinical symptom of the gastrointestinal disorder to be treated. In other embodiments, the effective amount is the 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. The therapeutic effects need not be complete or curative, as long as 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 among a mouse, a rat, a dog, a cat, a cow, a horse, or a pig.

0161The invention also provides methods for treating gastrointestinal cancer in a subject in need thereof by administering an effective amount of a GCC agonist or a formulation thereof to the subject. Non-limiting examples of gastrointestinal cancers that can be treated according to the methods of the invention include gastric cancer, esophageal cancer, pancreatic cancer, colorectal cancer, intestinal cancer, anal cancer, liver cancer, gallbladder cancer, or colon cancer.

0162The invention also provides methods for treating lipid metabolism disorders, biliary disorders, inflammatory disorders, lung disorders, cancer, cardiac disorders including cardiovascular disorders, eye disorders, oral disorders, blood disorders, liver disorders, skin disorders, prostate disorders, endocrine disorders, and obesity.

0163Lipid metabolism disorders include, but are not limited to, dyslipidemia, hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, sitosterolemia, familial hypercholesterolemia, xanthoma, combined hyperlipidemia, lecithin cholesterol acyltransferase deficiency, tangier disease, abetalipoproteinemia, erectile dysfunction, fatty liver disease, and hepatitis.

0164Biliary disorders include gallbladder disorders such as for example, gallstones, gall bladder cancer cholangitis, or primary sclerosing cholangitis; or bile duct disorders such as for example,cholecystitis, bile duct cancer or fascioliasis.

0165Inflammatory disorders include tissue and organ inflammation such as kidney inflammation (e.g., nephritis), gastrointestinal system inflammation (e.g., Crohn's disease and ulcerative colitis); necrotizing enterocolitis (NEC): pancreatic inflammation (e.g., pancreatis), pancreatic insufficiency, lung inflammation (e.g., bronchitis or asthma) or skin inflammation (e.g., psoriasis, eczema).

0166Lung Disorders include for example chronic obstructive pulmonary disease (COPD), and fibrosis.

0167Cancer includes tissue and organ carcinogenesis including metastases such as for example gastrointestinal cancer, ( e.g., gastric cancer, esophageal cancer, pancreatic cancer colorectal cancer, intestinal cancer, anal cancer, liver cancer, gallbladder cancer, or colon cancer; lung cancer; thyroid cancer; skin cancer (e.g., melanoma); oral cancer, urinary tract cancer (e.g. bladder cancer or kidney cancer); blood cancer (e.g. myeloma or leukemia) or prostate cancer.

0168Cardiac disorders include for example, congestive heart failure, trachea cardia hypertension, high cholesterol, or high triglycerides. Cardiovascular disorders include for example aneurysm, angina, atherosclerosis, cerebrovascular accident (stroke), cerebrovasculardisease, congestive heart failure, coronary artery disease, myocardial infarction (heart attack), or peripheral vascular disease.

0169Liver disorders include for example cirrhosis and fibrosis. In addition, GC-C agonist may also be useful to facilitate liver regeneration in liver transplant patients. Eye disorders include for example increased intra-ocular pressure, glaucoma, dry eyes, retinal degeneration, disorders of tear glands or eye inflammation. Skin disorders include for example xerosis. Oral disorders include for example dry mouth (xerostomia), Sjögren's syndrome, gum diseases (e.g., periodontal disease), or salivary gland duct blockage or malfunction. 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 Dosages

0170Disorders are treated, prevented or alleviated by administering to a subject, <i>e.g.,</i> a mammal such as a human in need thereof, a therapeutically effective dose of a GCC agonist peptide. The present invention is based in part on the unexpected results of clinical trials in humans which demonstrated that the formulations of the invention are therapeutically effective at much lower doses than predicted based on animal studies. In accordance with one aspect of the invention, the therapeutically effective dose is between 0.01 milligrams (mg) and 10 mg per unit dose. The term "unit dose" refers to a single drug delivery entity, <i>e.g.</i>, a tablet, capsule, solution, inhalation, controlled release or extended release formulation (<i>e.g.</i> MMX^® technology of Cosmo Pharmaceuticals). In one embodiment, the effective dose is between 0.01 mg and 9 mg. In another embodiment, the effective dose is between 0.01 mg and 5 mg. In another embodiment, the effective dose is between 0.01 mg and 3 mg. In another embodiment, the effective dose is between 0.10 mg and 5 mg. In another embodiment, the effective dose is between 0.10 mg and 3 mg. In one embodiment, the unit dose is .01 mg, .05 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.5 mg, 1.0 mg, 1.5 mg, 2.0 mg, 2.5 mg, 3.0 mg, 5 mg, or 10 mg. In one embodiment, the unit dose is 0.3 mg, 1.0 mg, 3.0 mg, 9.0 mg, or 9.5 mg.

0171The GCC agonist peptides may be in a pharmaceutical composition in unit dose form, together with one or more pharmaceutically acceptable excipients. The amount of peptide present should be sufficient to have a positive therapeutic effect when administered to a patient. What constitutes a "positive therapeutic effect" will depend upon the particular condition being treated and will include any significant improvement in a condition readily recognized by one of skill in the art.

0172The GCC agonists for use in the methods described above are preferably administered orally. Dosage forms include solutions, suspensions, emulsions, tablets, and capsules.

0173The total daily dose can be administered to the patient in a single dose, or in multiple sub-doses. Typically, sub-doses can 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.

0174The GCC agonists may be administered as either the sole active agent or in combination with one or more additional active agents. In all cases, additional active agents should be administered at a dosage that is therapeutically effective using the existing art as a guide. The GCC agonists may be administered in a single composition or sequentially with the one or more additional active agents. In one embodiment, the GCC agonist is administered in combination with one or more inhibitors of cGMP dependent phosphodiesterase such as suldinac sulfone, zaprinast, motapizone, vardenafil, or sildenifil. In another embodiment, the GCC agonist is administered in combination with one or more chemotherapeutic agents. In another embodiment, the GCC agonist is administered in combination with one or more or anti-inflammatory drugs such as steroids or non-steroidal anti-inflammatory drugs (NSAIDS), such as aspirin.

0175Combination therapy can be achieved by administering two or more agents, <i>e.g.,</i> a GCC agonist peptide described herein and another compound, each of which is formulated and administered separately, or by administering two or more agents in a single formulation. Other combinations are also encompassed by combination therapy. For example, two agents can be formulated together and administered in conjunction with a separate formulation containing a third agent. While the two or more agents in the combination therapy can be administered simultaneously, they need not be. For example, administration of a first agent (or combination of agents) can precede administration of a second agent (or combination of agents) by minutes, hours, days, or weeks. Thus, the two or more agents can 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 of each other or within 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks of each other. In some cases even longer intervals are possible. While in many cases it is desirable that the two or more agents used in a combination therapy be present in within the patient's body at the same time, this need not be so.

0176The GCC agonist peptides described herein may be combined with phosphodiesterase inhibitors, <i>e.g</i>., sulindae sulfone, Zaprinast, sildenafil, vardenafil or tadalafil to further enhance levels of cGMP in the target tissues or organs.

0177Combination therapy can also include two or more administrations of one or more of the agents used in the combination. For example, if agent X and agent Y are used in a combination, one could administer them sequentially in any combination one or more times, <i>e.g.,</i> in the order X-^Y- X, X-X-Y, Y-X-Y,Y-Y-X,X-X-Y-Y, etc.

1.2.2 Exemplary Agents for Combination Therapy

0178The 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 gastrointestinal disease or disorder. In some embodiments, the GCC agonist formulation comprises one or more additional therapeutic agents. In other embodiments, the GCC agonist is formulated separately from the one or more additional therapeutic agents. In accordance with this embodiment, the GCC agonist is administered either simultaneously, sequentially, or at a different time than the one or more additional therapeutic agents. In one embodiment, the GCC agonist formulation is administered in combination with one or more additional therapeutic agents selected from the group consisting of phosphodiesterase inhibitors, cyclic nucleotides (such as cGMP and cAMP), a laxative (such as SENNA or METAMUCIL), a stool softner, an anti-tumor necrosis factor alpha therapy for IBD (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 an inhibitor of cGMP-specific phosphodiesterase (cGMP-PDE) either concurrently or sequentially with said GCC agonist. cGMP-PDE inhibitors include, for example, suldinac sulfone, zaprinast, motapizone, vardenifil, and sildenafil. In another embodiment, the GCC agonist formulation is administered in combination with inhibitors of cyclic nucleotide transporters. Further examples of therapeutic agents that may be administered in combination with the GCC agonist formulations of the invention are given in the following sections.

1.2.2.1

Agents to Treat Gastrointestinal Cancers

0179The GCC agonist formulations described herein can be used in combination with one or more antitumor agents including but not limited to alkylating agents, epipodophyl lotoxitis, nitrosoureas, anti-metabolites, vinca alkaloids, anthracycline antibiotics, nitrogen mustard agents, and the like. Particular antitumor agents include tamoxifen, taxol, etoposide, and 5-fluorouracil. In one embodiment, the GCC agonist formulations are used in combination with an antiviral agent or a monoclonal antibody.

0180Non-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 anti-proliferative agents, agents for DNA modification or repair, DNA synthesis inhibitors, DNA/RNA transcription regulators, RNA processing inhibitors, agents that affect protein expression, synthesis and stability, agents that affect protein localization or their ability to exert their physiological action, agents that interfere with protein-protein or protein-nucleic acid interactions, agents that act by RNA interference, receptor binding molecules of any chemical nature (including small molecules and antibodies), targeted toxins, enzyme activators, enzyme inhibitors, gene regulators, HSP-90 inhibitors, molecules interfering with microtubules or other cytoskeletal components or cell adhesion and motility, agents for phototherapy, and therapy adjuncts.

0181Representative anti-proliferative agents include N-acetyl-D-sphingosine (C.sub.2 ceramide), apigenin, berberine chloride, dichloromethylenediphosphonic acid disodium salt, loe-emodine, emodin, HA 14-1, N-hexanoyl-D-sphingosine (C.sub.6 ceramide), 7b-hydroxycholesterol, 25-hydroxycholesterol, hyperforin, parthenolide, and rapamycin.

0182Representative agents for DNA modification and repair include aphidicolin, bleomycin sulfate, carboplatin, carmustine, chlorambucil, cyclophosphamide monohydrate, cyclophosphamide monohydrate ISOPAC.RTM., cis-diammineplatinum(II) dichloride (Cisplatin), esculetin, melphalan, methoxyamine hydrochloride, mitomycin C, mitoxantrone dihydrochloride, oxaliplatin, and streptozocin.

0183Representative DNA synthesis inhibitors include (.+-.)amethopterin (methotrexate), 3-amino-1,2,4-benzotriazine 1,4-dioxide, aminopterin, cytosine b-D-arabinofurdnoside (Ara-C), cytosine b-D-arabinofuranoside (Ara-C) hydrochloride, 2-fluoroadenine-9-b-D-arabinofuranoside (Fludarabine des-phosphate; F-ara-A), 5-fluoro-5'-deoxyuridinc, 5-fluorouracil, ganciclovir, hydroxyurea, 6-mercaptopurine, and 6-thioguanine.

0184Representative DNA/RNA transcription regulators include actinomycin D, daunorubicin hydrochloride, 5,6-dichlorobenzimidazole 1-b-D-ribofuranoside, doxorubicin hydrochloride, homoharringtonine, and idarubicin hydrochloride.

0185Representative enzyme activators and inhibitors include forskolin, DL-aminoglutethimide, apicidin, Bowman-Birk Inhibitor, butein, (S)-(+)-camptothecin, curcumin, (-)-deguelin, (-)-depudecin, doxycycline hyclate, etoposide, fonnestane, fostriecin sodium salt, hispidin, 2-imitio-1-imidazoliditicacetic acid (Cyclocreatine), oxamflatin, 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.

0186Representative gene regulators include 5-aza-2'-deoxycytidine, 5-azacytidine, cholecalciferol (Vitamin D3), ciglitizone, cyproterone acetate, 15-deoxy-D.sup.12,14-prostaglandin J.sub.2, epitestosterone, flutamide, glycyrrhizic acid ammonium salt (glycyrrhizin), 4-hydroxytamoxifen, mifepristone, procainamide hydrochloride, raloxifene hydrochloride, all trans-retinal (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 citrate salt, tetradecylthioacetic acid, and troglitazone.

0187Representative FiSP-90 inhibitors include 17-(allylamino)-17-demethoxygeldanamycin and geldanamycin.

0188Representative microtubule inhibitors include colchicines, dolastatin 15, nocodazole, taxanes and in particular paclitaxel, podophyllotoxin, rhizoxin, vinblastine sulfate salt, vincristine sulfate salt, and vindesine sulfate salt and vinorelbine (Navelbine) ditartrate salt.

0189Representative agents for performing phototherapy include photoactive porphyrin rings, hypericin, 5-methoxypsoralen, 8-methoxypsoralen, psoralen and ursodeoxycholic acid.

0190Representative agents used as therapy adjuncts include amifostine, 4-amino-1,8-naphthalimide, brefeldin A, cimetidine, phosphomycin disodium salt, leuprolide (leuprorelin) acetate salt, luteinizing hormone-releasing hormone (LH-RH) acetate salt, lectin, papaverine hydrochloride, pifithrin-a, (-)-scopolamine hydrobromide, and thapsigargin.

0191The agents can also be anti-VEGF (vascular endothelial growth factor) agents, as such are known in the art. Several antibodies and small molecules are currently in clinical trials or have been approved that function by inhibiting VEGF, such as Avastin (Bevacizumab), SU5416, SU11248 and BAY 43-9006. The agents can also be directed against growth factor receptors such as those of the EGF/Erb-B family such as EGF Receptor (Iressa or Gefitinib, and Tarceva or Erlotinib), Erb-B2, receptor (Herceptin or Trastuzumab), other receptors (such as Rituximab or Rituxan/MabThera), tyrosine kinases, non-receptor tyrosine kinases, cellular serine/threonine kinases (including MAP kinases), and various other proteins whose deregulation contribute to oncogenesis (such as small/Ras family and large/heterotrimeric G proteins). Several antibodies and small molecules targeting those molecules are currently at various stages of development (including approved for treatment or in clinical trials).

0192In a preferred embodiment, the invention provides a method for treating colon cancer in a subject in need thereof by administering to the subject a GCC, agonist formulation in combination with one or more antitumor agent selected from the group consisting of paclitaxel, docetaxel, tamoxifen, vinorelbine, gemcitabine, cisplatin, etoposide, topotecan, irinotecan, anastrozole, rituximab, trastuzumab, fludarabine, cyclophosphamide, gentuzumab, carboplatin, interferons, and doxorubicin. In a particular embodiment the antitumor agent is paclitaxel. In a further embodiment, the method further comprises an antitumor agent selected from the group consisting of 5-FU, doxorubicin, vinorelbine, cytoxan, and cisplatin.

1.2.2.2

Agents that Treat Crohn's Disease

0193In one embodiment, a GCC agonist formulation of the invention is administered as part of a combination therapy with one or more additional therapeutic agents for the treatment of Crohn's disease. Non-limiting examples of the one or more additional therapeutic agents include sulfasalazine and other mesalamine-containing drugs, generally known as 5-ASA agents, such as Asacol, Dipentum, or Pentasa, or infliximab (REMICADE). In certain embodiments, the one or more additional agents is a corticosteroid or an immunosuppressive agent such as 6-mercaptopurine or azathioprine. In another embodiment, the one or more additional agents is an antidiarrheal agent such as diphenoxylate, loperamide, or codeine.

1.2.2.3

Agents that Treat Ulcerative Colitis

0194In one embodiment, a GCC agonist formulation of the invention is administered as part of a combination therapy with one or more additional therapeutic agents for the treatment of ulcerative colitis. The agents that are used to treat ulcerative colitis overlap with those used to treat Chrohn's Disease. Non-limiting examples of the one or more additional therapeutic agents that can be used in combination with a GCC, agonist formulation of the invention include aminosalicylates (drugs that contain 5-aminosalicyclic acid (5-ASA)) such as sulfasalazine, olsalazine, mesalamine, and balsalazide. Other therapeutic agents that can be used include corticosteroids, such as prednisone and hydrocortisone, immunomodulators, such as azathioprine, 6-mercapto-purine (6-MP), cytokines, interleukins, and lymphokines, and anti-TNF-alpha agents, including the thiazolidinediones or glitazones such as rosiglitazone and pioglitazone. In one emobidment, the one or more additional therapeutic agents includes both cyclosporine A and 6-MP or azathioprine for the treatment of active, severe ulcerative colitis.

1.2.2.4

Agents that Treat Constipation/Irritable Dowel Syndrome

0195In one embodiment, a GCC agonist formulation of the invention is administered as part of a combination therapy with one or more additional therapeutic agents for the treatment of constipation, such as that associated with irritable bowel syndrome. Non-limiting examples of the one or more additional therapeutic agents include laxatives such as SENNA, MIRALAX, LACTULOSE, PEG, or calcium polycarbophil), stool softeners (such as mineral oil or COLACE), bulking agents (such as METAMUCIL or bran), agents such as ZELNORM (also called tegaserod), and anticholinergic medications such as BENTYL and LEVSIN.

1.2.2.5

Agents for the Treatment of Postoperative Ileus

0196In one embodiment, a GCC agonist formulation of the invention is administered as part of a combination therapy with one or more additional therapeutic agents for the treatment of postoperative ileus. Non-limiting examples of the one or more additional therapeutic agents include ENTEREG (alvimopan; formerly called ado lor! ADL 8-2698), conivaptan, and related agents describes in <patcit id="pcit0032" dnum="US6645959B"><text>US 6,645,959</text></patcit>.

1.2.2.6

Anti-obesity agents

0197In one embodiment, a GCC agonist formulation of the invention is administered as part of a combination therapy with one or more additional therapeutic agents for the treatment of obesity. Non-limiting examples of the one or more additional therapeutic agents include 1 1β HSD-1 (11-beta hydroxy steroid dehydrogenase type 1) inhibitors, such as BVT 3498, BVT 2733, 3-(1-adamantyl)-4-ethyl-5-(ethylthio)- 4H-l,2,4-triazole, 3-(1-adamantyl)-5-(3,4,5-trimethoxypheny1)-4-methyl-4H-l,2,4-triazole, 3- adamantanyl-4,5,6,7,8,9,10,11,12,3a-decahydro-l,2,4-triazolo[4,3-a][l l]annulene, and those compounds disclosed in <patcit id="pcit0033" dnum="WO0190091A"><text>WO01/90091</text></patcit>, <patcit id="pcit0034" dnum="WOO190090A"><text>WOO 1/90090</text></patcit>, <patcit id="pcit0035" dnum="WOO190092A"><text>WOO 1/90092</text></patcit> and <patcit id="pcit0036" dnum="WO02072084A"><text>WO02/072084</text></patcit>; 5HT antagonists such as those in_ <patcit id="pcit0037" dnum="WO03037871A"><text>WO03/037871</text></patcit>, <patcit id="pcit0038" dnum="WO03037887A"><text>WO03/037887</text></patcit>, and the like; 5HTIa modulators such as carbidopa, benserazide and those disclosed in <patcit id="pcit0039" dnum="US6207699B"><text>US6207699</text></patcit>, <patcit id="pcit0040" dnum="WO03031439A"><text>WO03/031439</text></patcit>, and the like; 5HT2c (serotonin receptor 2c) agonists, such as BVT933, DPCA37215, IK264, PNU 22394, WAY161503, R-1065, SB 243213 (Glaxo Smith Kline) and YM 348 and those disclosed in <patcit id="pcit0041" dnum="US3914250A"><text>US3914250</text></patcit>, <patcit id="pcit0042" dnum="WO0077010A"><text>WO00/77010</text></patcit>, <patcit id="pcit0043" dnum="WO0236596A"><text>WO02/36596</text></patcit>, <patcit id="pcit0044" dnum="WO0248124A"><text>WO02/48124</text></patcit>, <patcit id="pcit0045" dnum="WO0210169A"><text>WO02/10169</text></patcit>, <patcit id="pcit0046" dnum="WO0166548A"><text>WO01/66548</text></patcit>, <patcit id="pcit0047" dnum="WO0244152A"><text>WO02/44152</text></patcit>, <patcit id="pcit0048" dnum="WO0251844A"><text>WO02/51844</text></patcit>, <patcit id="pcit0049" dnum="WO0240456A"><text>WO02/40456</text></patcit>, and <patcit id="pcit0050" dnum="WO0240457A"><text>WO02/40457</text></patcit>; 51-IT6 receptor modulators, such as those in <patcit id="pcit0051" dnum="WO03030901A"><text>WO03/030901</text></patcit>, <patcit id="pcit0052" dnum="WO03035061A"><text>WO03/035061</text></patcit>, <patcit id="pcit0053" dnum="WO03039547A"><text>WO03/039547</text></patcit>, and the like; acyl-estrogens, such as oleoyl-estrone, disclosed in <nplcit id="ncit0023" npl-type="s"><text>del Mar-Grasa, M. et al, Obesity Research, 9:202-9 (2001</text></nplcit>) and Japanese Patent Application No. <patcit id="pcit0054" dnum="JP2000256190B"><text>JP 2000256190</text></patcit>; anorectic bicyclic compounds such as 1426 (Aventis) and 1954 (Aventis), and the compounds disclosed in <patcit id="pcit0055" dnum="WO0018749A"><text>WO00/18749</text></patcit>, <patcit id="pcit0056" dnum="WO0132638A"><text>WO01/32638</text></patcit>, <patcit id="pcit0057" dnum="WO0162746A"><text>WO01/62746</text></patcit>, <patcit id="pcit0058" dnum="WO0162747A"><text>WO01/62747</text></patcit>, and <patcit id="pcit0059" dnum="WO03015769A"><text>WO03/015769</text></patcit>; CB 1 (cannabinoid-1 receptor) antagonist/inverse agonists such as rimonabant (Acomplia; Sanofi), SR-14778 (Sanofi), SR-141716 (Sanofi), BAY 65-2520 (Bayer), and SLV 319 (Solvay), and those disclosed in patent publications <patcit id="pcit0060" dnum="US4973587A"><text>US4973587</text></patcit>, <patcit id="pcit0061" dnum="US5013837A"><text>US5013837</text></patcit>, <patcit id="pcit0062" dnum="US5081122A"><text>US5081122</text></patcit>, <patcit id="pcit0063" dnum="US5112820A"><text>US5112820</text></patcit>, <patcit id="pcit0064" dnum="US5292736A"><text>US5292736</text></patcit>, <patcit id="pcit0065" dnum="US5532237A"><text>US5532237</text></patcit>, <patcit id="pcit0066" dnum="US5624941A"><text>US5624941</text></patcit>, <patcit id="pcit0067" dnum="US6028084A"><text>US6028084</text></patcit>, <patcit id="pcit0068" dnum="US6509367B"><text>US6509367</text></patcit>, <patcit id="pcit0069" dnum="US6509367B"><text>US6509367</text></patcit>, <patcit id="pcit0070" dnum="WO9633159A"><text>WO96/33159</text></patcit>, <patcit id="pcit0071" dnum="WO9729079A"><text>WO97/29079</text></patcit>, <patcit id="pcit0072" dnum="WO9831227A"><text>WO98/31227</text></patcit>, <patcit id="pcit0073" dnum="WO9833765A"><text>WO98/33765</text></patcit>, <patcit id="pcit0074" dnum="WO9837061A"><text>WO98/37061</text></patcit>, <patcit id="pcit0075" dnum="WO9841519A"><text>WO98/41519</text></patcit>, <patcit id="pcit0076" dnum="WO9843635A"><text>WO98/43635</text></patcit>, <patcit id="pcit0077" dnum="WO9843636A"><text>WO98/43636</text></patcit>, <patcit id="pcit0078" dnum="WO9902499A"><text>WO99/02499</text></patcit>, <patcit id="pcit0079" dnum="WO0010967A"><text>WO00/10967</text></patcit>, <patcit id="pcit0080" dnum="WO0010968A"><text>WO00/10968</text></patcit>, <patcit id="pcit0081" dnum="WO0109120A"><text>WO01/09120</text></patcit>, <patcit id="pcit0082" dnum="WO0158869A"><text>WO01/58869</text></patcit>, <patcit id="pcit0083" dnum="WO0164632A"><text>WO01/64632</text></patcit>, <patcit id="pcit0084" dnum="WO0164633A"><text>WO01/64633</text></patcit>, <patcit id="pcit0085" dnum="WO0164634A"><text>WO01/64634</text></patcit>, <patcit id="pcit0086" dnum="WO0170700A"><text>WO01/70700</text></patcit>, <patcit id="pcit0087" dnum="WO0196330A"><text>WO01/96330</text></patcit>, <patcit id="pcit0088" dnum="WO02076949A"><text>WO02/076949</text></patcit>, <patcit id="pcit0089" dnum="WO03006007A"><text>WO03/006007</text></patcit>, <patcit id="pcit0090" dnum="WO03007887A"><text>WO03/007887</text></patcit>, <patcit id="pcit0091" dnum="WO03020217A"><text>WO03/020217</text></patcit>, <patcit id="pcit0092" dnum="WO03026647A"><text>WO03/026647</text></patcit>, <patcit id="pcit0093" dnum="WO03026648A"><text>WO03/026648</text></patcit>, <patcit id="pcit0094" dnum="WO03027069A"><text>WO03/027069</text></patcit>, <patcit id="pcit0095" dnum="WO03027076A"><text>WO03/027076</text></patcit>, <patcit id="pcit0096" dnum="WO03027114A"><text>WO03/027114</text></patcit>, <patcit id="pcit0097" dnum="WO03037332A"><text>WO03/037332</text></patcit>, <patcit id="pcit0098" dnum="WO03040107A"><text>WO03/040107</text></patcit>, <patcit id="pcit0099" dnum="WO03086940A"><text>WO03/086940</text></patcit>, <patcit id="pcit0100" dnum="WO03084943A"><text>WO03/084943</text></patcit> and <patcit id="pcit0101" dnum="EP658546A"><text>EP658546</text></patcit>; CCK-A (cholecystokinin-A) agonists, such as AP,-R 15849, GI 181771 (GSK), JMV-180, A- 71378, A-71623 and SR146131 (Sanofi), and those described in <patcit id="pcit0102" dnum="US5739106A"><text>US5739106</text></patcit>; CNTF (Ciliary neurotrophic factors), such as GI- 181771 (Glaxo-SmithKline), SRl 46131 (Sanofi Synthelabo), butabindide, PD 170,292, and PD 149164 (Pfizer); CNTF derivatives, such as Axokine^® (Regeneron), and those disclosed in <patcit id="pcit0103" dnum="WO9409134A"><text>WO94/09134</text></patcit>, <patcit id="pcit0104" dnum="WO9822128A"><text>WO98/22128</text></patcit>, and <patcit id="pcit0105" dnum="WO9943813A"><text>WO99/43813</text></patcit>; dipeptidyl peptidase IV (DP-IV) inhibitors, such as isoleucine thiazolidide, valine pyrrolidide, NVP-DPP728, LAF237, P93/01, P 3298, TSL 225 (tryptophyl-l,2,3,4-tetrahydroisoquinoline-3- carboxylic acid; disclosed by <nplcit id="ncit0024" npl-type="s"><text>Yamada et al, Bioorg. &amp; Med. Chem. Lett. 8 (1998) 1537-1540</text></nplcit>), TMC-2A/2B/2C, CD26 inhibtors, FE 999011, P9310/K364, VIP 0177, SDZ 274-444, 2- cyanopyrrolidides and 4-cyanopyrrolidides as disclosed by <nplcit id="ncit0025" npl-type="s"><text>Ashworth et al, Bioorg. &amp; Med. Chem. Lett., Vol. 6, No. 22, pp 1163-1166 and 2745-2748 (1996</text></nplcit>) and the compounds disclosed patent publications. <patcit id="pcit0106" dnum="WO9938501A"><text>WO99/38501</text></patcit>, <patcit id="pcit0107" dnum="WO9946272A"><text>WO99/46272</text></patcit>, <patcit id="pcit0108" dnum="WO9967279A"><text>WO99/67279 (Probiodrug</text></patcit>), <patcit id="pcit0109" dnum="WO9967278A"><text>WO99/67278 (Probiodrug</text></patcit>), <patcit id="pcit0110" dnum="WO9961431A"><text>WO99/61431 (Probiodrug</text></patcit>), <patcit id="pcit0111" dnum="WO02083128A"><text>WO02/083128</text></patcit>, <patcit id="pcit0112" dnum="WO02062764A"><text>WO02/062764</text></patcit>, <patcit id="pcit0113" dnum="WO03000180A"><text>WO03/000180</text></patcit>, <patcit id="pcit0114" dnum="WO03000181A"><text>WO03/000181</text></patcit>, <patcit id="pcit0115" dnum="WO03000250A"><text>WO03/000250</text></patcit>, <patcit id="pcit0116" dnum="WO03002530A"><text>WO03/002530</text></patcit>, <patcit id="pcit0117" dnum="WO03002531A"><text>WO03/002531</text></patcit>, <patcit id="pcit0118" dnum="WO03002553A"><text>WO03/002553</text></patcit>, <patcit id="pcit0119" dnum="WO03002593A"><text>WO03/002593</text></patcit>, <patcit id="pcit0120" dnum="WO03004498A"><text>WO03/004498</text></patcit>, <patcit id="pcit0121" dnum="WO03004496A"><text>WO03/004496</text></patcit>, <patcit id="pcit0122" dnum="WO03017936A"><text>WO03/017936</text></patcit>, <patcit id="pcit0123" dnum="WO03024942A"><text>WO03/024942</text></patcit>, <patcit id="pcit0124" dnum="WO03024965A"><text>WO03/024965</text></patcit>, <patcit id="pcit0125" dnum="WO03033524A"><text>WO03/033524</text></patcit>, <patcit id="pcit0126" dnum="WO03037327A"><text>WO03/037327</text></patcit> and <patcit id="pcit0127" dnum="EP1258476A"><text>EP1258476</text></patcit>; 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 disclosed in <patcit id="pcit0128" dnum="US662448"><text>USSN 09/662448</text></patcit>, <patcit id="pcit0129" dnum="US203335"><text>US provisional application 60/203335</text></patcit>, <patcit id="pcit0130" dnum="US6358951B"><text>US6358951</text></patcit>, <patcit id="pcit0131" dnum="US2002049196A"><text>US2002049196</text></patcit>, <patcit id="pcit0132" dnum="US2002022637A"><text>US2002/022637</text></patcit>, <patcit id="pcit0133" dnum="WO0156592A"><text>WO01/56592</text></patcit> and <patcit id="pcit0134" dnum="WO0232888A"><text>WO02/32888</text></patcit>; H3 (histamine H3) antagonist/inverse agonists, such as thioperamide, 3-(1H-imidazol-4- yl)propyl N-(4-pentenyl)carbamate), clobenpropit, iodophenpropit, imoproxifan, GT2394 (Gliatech), and A331440, O-[3-(1H-imidazol-4-yl)propanol]carbamates (<nplcit id="ncit0026" npl-type="s"><text>Kiec-Kononowicz, K. et al., Pharmazie, 55:349-55 (2000</text></nplcit>)), piperidine-containing histamine H3-receptor antagonists (<nplcit id="ncit0027" npl-type="s"><text>Lazewska, D. et al., Pharmazie, 56:927-32 (2001</text></nplcit>), benzophenone derivatives and related compounds (<nplcit id="ncit0028" npl-type="s"><text>Sasse, A. et al., Arch. Pharm.(Weinheim) 334:45-52 (2001</text></nplcit>)), substituted N- phenylcarbamates (<nplcit id="ncit0029" npl-type="s"><text>Reidemeister, S. et al., Pharmazie, 55:83-6 (2000</text></nplcit>)), and proxifan derivatives (<nplcit id="ncit0030" npl-type="s"><text>Sasse, A. et al., J. Med. Chem. 43:3335-43 (2000</text></nplcit>)) and histamine H3 receptor modulators such as those disclosed in <patcit id="pcit0135" dnum="WO0215905A"><text>WO02/15905</text></patcit>, <patcit id="pcit0136" dnum="WO03024928A"><text>WO03/024928</text></patcit> and <patcit id="pcit0137" dnum="WO03024929A"><text>WO03/024929</text></patcit>; leptin derivatives, such as those disclosed in <patcit id="pcit0138" dnum="US5552524A"><text>US5552524</text></patcit>, <patcit id="pcit0139" dnum="US5552523A"><text>US5552523</text></patcit>, <patcit id="pcit0140" dnum="US5552522A"><text>US5552522</text></patcit>, <patcit id="pcit0141" dnum="US5521283A"><text>US5521283</text></patcit>, <patcit id="pcit0142" dnum="WO9623513A"><text>WO96/23513</text></patcit>, <patcit id="pcit0143" dnum="WO9623514A"><text>WO96/23514</text></patcit>, <patcit id="pcit0144" dnum="WO9623515A"><text>WO96/23515</text></patcit>, <patcit id="pcit0145" dnum="WO963516A"><text>WO96/3516</text></patcit>, <patcit id="pcit0146" dnum="WO9623517A"><text>WO96/23517</text></patcit>, <patcit id="pcit0147" dnum="WO9623518A"><text>WO96/23518</text></patcit>, <patcit id="pcit0148" dnum="WO9623519A"><text>WO96/23519</text></patcit>, and <patcit id="pcit0149" dnum="WO9623520A"><text>WO96/23520</text></patcit>; leptin, including recombinant human leptin (PEG-OB, Hoffman La Roche) and recombinant methionyl human leptin (Amgen); lipase inhibitors, such as tetrahydrolipstatin (orlistat/Xenical^®), Triton WRl 339, RHC80267, lipstatin, teasaponin, diethylumbelliferyl phosphate, FL-386, WAY-121898, Bay-N-3176, valilactone, esteracin, ebelactone A, ebelactone B, and RHC 80267, and those disclosed in patent publications <patcit id="pcit0150" dnum="WO0177094A"><text>WO01/77094</text></patcit>, <patcit id="pcit0151" dnum="US4598089A"><text>US4598089</text></patcit>, <patcit id="pcit0152" dnum="US4452813A"><text>US4452813</text></patcit>,<patcit id="pcit0153" dnum="US5512565A"><text> USUS5512565</text></patcit>, <patcit id="pcit0154" dnum="US5391571A"><text>US5391571</text></patcit>, <patcit id="pcit0155" dnum="US5602151A"><text>US5602151</text></patcit>, <patcit id="pcit0156" dnum="US4405644A"><text>US4405644</text></patcit>, <patcit id="pcit0157" dnum="US4189438A"><text>US4189438</text></patcit>, and <patcit id="pcit0158" dnum="US4242453A"><text>US4242453</text></patcit>; lipid metabolism modulators such as maslinic acid, erythrodiol, ursolic acid uvaol, betulinic acid, betulin, and the like and compounds disclosed in <patcit id="pcit0159" dnum="WO03011267A"><text>WO03/011267</text></patcit>; Mc4r (melanocortin 4 receptor) agonists, such as CHIR86036 (Chiron), ME- 10142, ME-10145, and HS-131 (Melacure), and those disclosed in <patcit id="pcit0160" dnum="WO9964002A"><text>PCT publication Nos. WO99/64002</text></patcit>, <patcit id="pcit0161" dnum="WO0074679A"><text>WO00/74679</text></patcit>, <patcit id="pcit0162" dnum="WOO1991752A"><text>WOO 1/991752</text></patcit>, <patcit id="pcit0163" dnum="WOO125192A"><text>WOO 1/25192</text></patcit>, <patcit id="pcit0164" dnum="WOO152880A"><text>WOO 1/52880</text></patcit>, <patcit id="pcit0165" dnum="WOO174844A"><text>WOO 1/74844</text></patcit>, <patcit id="pcit0166" dnum="WOO170708A"><text>WOO 1/70708</text></patcit>, <patcit id="pcit0167" dnum="WO0170337A"><text>WO01/70337</text></patcit>, <patcit id="pcit0168" dnum="WO0191752A"><text>WO01/91752</text></patcit>, <patcit id="pcit0169" dnum="WO02059095A"><text>WO02/059095</text></patcit>, <patcit id="pcit0170" dnum="WO02059107A"><text>WO02/059107</text></patcit>, <patcit id="pcit0171" dnum="WO02059108A"><text>WO02/059108</text></patcit>, <patcit id="pcit0172" dnum="WO021059117A"><text>WO021/059117</text></patcit>, <patcit id="pcit0173" dnum="WO0206276A"><text>WO02/06276</text></patcit>, <patcit id="pcit0174" dnum="WO0212166A"><text>WO02/12166</text></patcit>, <patcit id="pcit0175" dnum="WO0211715A"><text>WO02/11715</text></patcit>, <patcit id="pcit0176" dnum="WO0212178A"><text>WO02/12178</text></patcit>, <patcit id="pcit0177" dnum="WO0215909A"><text>WO02/15909</text></patcit>, <patcit id="pcit0178" dnum="WO0238544A"><text>WO02/38544</text></patcit>, <patcit id="pcit0179" dnum="WO02068387A"><text>WO02/068387</text></patcit>, <patcit id="pcit0180" dnum="WO02068388A"><text>WO02/068388</text></patcit>, <patcit id="pcit0181" dnum="WO02067869A"><text>WO02/067869</text></patcit>, <patcit id="pcit0182" dnum="WO02081430A"><text>WO02/081430</text></patcit>, <patcit id="pcit0183" dnum="WO0306604A"><text>WO03/06604</text></patcit>, <patcit id="pcit0184" dnum="WO03007949A"><text>WO03/007949</text></patcit>, <patcit id="pcit0185" dnum="WO03009847A"><text>WO03/009847</text></patcit>, <patcit id="pcit0186" dnum="WO03009850A"><text>WO03/009850</text></patcit>, <patcit id="pcit0187" dnum="WO03013509A"><text>WO03/013509</text></patcit>, and <patcit id="pcit0188" dnum="WO03031410A"><text>WO03/031410</text></patcit>; Mc5r (melanocortin 5 receptor) modulators, such as those disclosed in <patcit id="pcit0189" dnum="WO9719952A"><text>WO97/19952</text></patcit>, <patcit id="pcit0190" dnum="WO0015826A"><text>WO00/15826</text></patcit>, <patcit id="pcit0191" dnum="WO0015790A"><text>WO00/15790</text></patcit>, <patcit id="pcit0192" dnum="US20030092041A"><text>US20030092041</text></patcit>; melanin-concentrating hormone 1 receptor (MCHR) antagonists, such as T-226296 (Takeda), SB 568849, SNP-7941 (Synaptic), and those disclosed in patent publications <patcit id="pcit0193" dnum="WOO121169A"><text>WOO 1/21169</text></patcit>, <patcit id="pcit0194" dnum="WO0182925A"><text>WO01/82925</text></patcit>, <patcit id="pcit0195" dnum="WO0187834A"><text>WO01/87834</text></patcit>, <patcit id="pcit0196" dnum="WO02051809A"><text>WO02/051809</text></patcit>, <patcit id="pcit0197" dnum="WO0206245A"><text>WO02/06245</text></patcit>, <patcit id="pcit0198" dnum="WO02076929A"><text>WO02/076929</text></patcit>, <patcit id="pcit0199" dnum="WO02076947A"><text>WO02/076947</text></patcit>, <patcit id="pcit0200" dnum="WO0204433A"><text>WO02/04433</text></patcit>, <patcit id="pcit0201" dnum="WO0251809A"><text>WO02/51809</text></patcit>, <patcit id="pcit0202" dnum="WO02083134A"><text>WO02/083134</text></patcit>, <patcit id="pcit0203" dnum="WO02094799A"><text>WO02/094799</text></patcit>, <patcit id="pcit0204" dnum="WO03004027A"><text>WO03/004027</text></patcit>, <patcit id="pcit0205" dnum="WO0313574A"><text>WO03/13574</text></patcit>, <patcit id="pcit0206" dnum="WO0315769A"><text>WO03/15769</text></patcit>, <patcit id="pcit0207" dnum="WO03028641A"><text>WO03/028641</text></patcit>, <patcit id="pcit0208" dnum="WO03035624A"><text>WO03/035624</text></patcit>, <patcit id="pcit0209" dnum="WO03033476A"><text>WO03/033476</text></patcit>, <patcit id="pcit0210" dnum="WO03033480A"><text>WO03/033480</text></patcit>, <patcit id="pcit0211" dnum="JP13226269B"><text>JP13226269</text></patcit>, and <patcit id="pcit0212" dnum="JP1437059A"><text>JP1437059</text></patcit>; mGluR5 modulators such as those disclosed in <patcit id="pcit0213" dnum="WO03029210A"><text>WO03/029210</text></patcit>, <patcit id="pcit0214" dnum="WO03047581A"><text>WO03/047581</text></patcit>, <patcit id="pcit0215" dnum="WO03048137A"><text>WO03/048137</text></patcit>, <patcit id="pcit0216" dnum="WO03051315A"><text>WO03/051315</text></patcit>, <patcit id="pcit0217" dnum="WO03051833A"><text>WO03/051833</text></patcit>, <patcit id="pcit0218" dnum="WO03053922A"><text>WO03/053922</text></patcit>, <patcit id="pcit0219" dnum="WO03059904A"><text>WO03/059904</text></patcit>, and the like; serotoninergic agents, such as fenfluramine (such as Pondimin^® (Benzeneethanamine, N-ethyl- alpha-methyl-3-(trifluoromethyl)-, hydrochloride), Robbins), dexfenfluramine (such as Redux^® (Benzeneethanamine, N-ethyl-alpha-methyl-3-(trifluoromethyl)-, hydrochloride), Interneuron) and sibutramine ((Meridia^®, Knoll/Reductil^™) including racemic mixtures, as optically pure isomers (+) and (-), and pharmaceutically acceptable salts, solvents, hydrates, clathrates and prodrugs thereof including sibutramine hydrochloride monohydrate salts thereof, and those compounds disclosed in <patcit id="pcit0220" dnum="US4746680A"><text>US4746680</text></patcit>, <patcit id="pcit0221" dnum="US4806570A"><text>US4806570</text></patcit>, and <patcit id="pcit0222" dnum="US5436272A"><text>US5436272</text></patcit>, <patcit id="pcit0223" dnum="US20020006964A"><text>US20020006964</text></patcit>, <patcit id="pcit0224" dnum="WOO127068A"><text>WOO 1/27068</text></patcit>, and <patcit id="pcit0225" dnum="WOO162341A"><text>WOO 1/62341</text></patcit>; NE (norepinephrine) transport inhibitors, such as GW 320659, despiramine, talsupram, and nomifensine; NPY 1 antagonists, such as BIBP3226, J-115814, BIBO 3304, LY-357897, CP-671906, GI- 264879A, and those disclosed in <patcit id="pcit0226" dnum="US6001836A"><text>US6001836</text></patcit>, <patcit id="pcit0227" dnum="WO96114307A"><text>WO961/14307</text></patcit>, <patcit id="pcit0228" dnum="WO0123387A"><text>WO01/23387</text></patcit>, <patcit id="pcit0229" dnum="WO9951600A"><text>WO99/51600</text></patcit>, <patcit id="pcit0230" dnum="WO0185690A"><text>WO01/85690</text></patcit>, <patcit id="pcit0231" dnum="WO0185098A"><text>WO01/85098</text></patcit>, <patcit id="pcit0232" dnum="WO0185173A"><text>WO01/85173</text></patcit>, and <patcit id="pcit0233" dnum="WO0189528A"><text>WO01/89528</text></patcit>; NPY5 (neuropeptide Y Y5) antagonists, such as 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, JCF-104, and H409/22 and those compounds disclosed in patent publications <patcit id="pcit0234" dnum="US6140354A"><text>US6140354</text></patcit>, <patcit id="pcit0235" dnum="US6191160B"><text>US6191160</text></patcit>, <patcit id="pcit0236" dnum="US6218408B"><text>US6218408</text></patcit>, <patcit id="pcit0237" dnum="US6258837B"><text>US6258837</text></patcit>, <patcit id="pcit0238" dnum="US6313298B"><text>US6313298</text></patcit>, <patcit id="pcit0239" dnum="US6326375B"><text>US6326375</text></patcit>, <patcit id="pcit0240" dnum="US6329395B"><text>US6329395</text></patcit>, <patcit id="pcit0241" dnum="US6335345B"><text>US6335345</text></patcit>, <patcit id="pcit0242" dnum="US6337332B"><text>US6337332</text></patcit>, <patcit id="pcit0243" dnum="US6329395B"><text>US6329395</text></patcit>, <patcit id="pcit0244" dnum="US6340683B"><text>US6340683</text></patcit>, <patcit id="pcit0245" dnum="EP01010691A"><text>EP01010691</text></patcit>, <patcit id="pcit0246" dnum="EP01044970A"><text>EP-01044970</text></patcit>, <patcit id="pcit0247" dnum="WO9719682A"><text>WO97/19682</text></patcit>, <patcit id="pcit0248" dnum="WO9720820A"><text>WO97/20820</text></patcit>, <patcit id="pcit0249" dnum="WO9720821A"><text>WO97/20821</text></patcit>, <patcit id="pcit0250" dnum="WO9720822A"><text>WO97/20822</text></patcit>, <patcit id="pcit0251" dnum="WO9720823A"><text>WO97/20823</text></patcit>, <patcit id="pcit0252" dnum="WO9827063A"><text>WO98/27063</text></patcit>, <patcit id="pcit0253" dnum="WO00107409A"><text>WO00/107409</text></patcit>, <patcit id="pcit0254" dnum="WO00185714A"><text>WO00/185714</text></patcit>, <patcit id="pcit0255" dnum="WO00185730A"><text>WO00/185730</text></patcit>, <patcit id="pcit0256" dnum="WO0064880A"><text>WO00/64880</text></patcit>, <patcit id="pcit0257" dnum="WO0068197A"><text>WO00/68197</text></patcit>, <patcit id="pcit0258" dnum="WO0069849A"><text>WO00/69849</text></patcit>, <patcit id="pcit0259" dnum="WO0113917A"><text>WO/0113917</text></patcit>, <patcit id="pcit0260" dnum="WO0109120A"><text>WO01/09120</text></patcit>, <patcit id="pcit0261" dnum="WO0114376A"><text>WO01/14376</text></patcit>, <patcit id="pcit0262" dnum="WO0185714A"><text>WO01/85714</text></patcit>, <patcit id="pcit0263" dnum="WO0185730A"><text>WO01/85730</text></patcit>, <patcit id="pcit0264" dnum="WO0107409A"><text>WO01/07409</text></patcit>, <patcit id="pcit0265" dnum="WO0102379A"><text>WO01/02379</text></patcit>, <patcit id="pcit0266" dnum="WO0123388A"><text>WO01/23388</text></patcit>, <patcit id="pcit0267" dnum="WO0123389A"><text>WO01/23389</text></patcit>, <patcit id="pcit0268" dnum="WOO144201A"><text>WOO 1/44201</text></patcit>, <patcit id="pcit0269" dnum="WO0162737A"><text>WO01/62737</text></patcit>, <patcit id="pcit0270" dnum="WO0162738A"><text>WO01/62738</text></patcit>, <patcit id="pcit0271" dnum="WO0109120A"><text>WO01/09120</text></patcit>, <patcit id="pcit0272" dnum="WO0220488A"><text>WO02/20488</text></patcit>, <patcit id="pcit0273" dnum="WO0222592A"><text>WO02/22592</text></patcit>, <patcit id="pcit0274" dnum="WO0248152A"><text>WO02/48152</text></patcit>, <patcit id="pcit0275" dnum="WO0249648A"><text>WO02/49648</text></patcit>, <patcit id="pcit0276" dnum="WO02051806A"><text>WO02/051806</text></patcit>, <patcit id="pcit0277" dnum="WO02094789A"><text>WO02/094789</text></patcit>, <patcit id="pcit0278" dnum="WO03009845A"><text>WO03/009845</text></patcit>, <patcit id="pcit0279" dnum="WO03014083A"><text>WO03/014083</text></patcit>, <patcit id="pcit0280" dnum="WO03022849A"><text>WO03/022849</text></patcit>, <patcit id="pcit0281" dnum="WO03028726A"><text>WO03/028726</text></patcit> and <nplcit id="ncit0031" npl-type="s"><text>Norman et al, J. Med. Chem. 43:4288-4312 (2000</text></nplcit>); opioid antagonists, such as nalmefene (PEVEX ^®), 3-methoxynaltrexone, methylnaltrexone, naloxone, and naltrexone (e.g. PT901; Pain Therapeutics, Inc.) and those disclosed in <patcit id="pcit0282" dnum="US20050004155A"><text>US20050004155</text></patcit> and <patcit id="pcit0283" dnum="WO0021509A"><text>WO00/21509</text></patcit>; orexin antagonists, such as SB-334867-A and those disclosed in patent publications <patcit id="pcit0284" dnum="WO0196302A"><text>WO01/96302</text></patcit>, <patcit id="pcit0285" dnum="WO0168609A"><text>WO01/68609</text></patcit>, <patcit id="pcit0286" dnum="WO0244172A"><text>WO02/44172</text></patcit>, <patcit id="pcit0287" dnum="WO0251232A"><text>WO02/51232</text></patcit>, <patcit id="pcit0288" dnum="WO0251838A"><text>WO02/51838</text></patcit>, <patcit id="pcit0289" dnum="WO02089800A"><text>WO02/089800</text></patcit>, <patcit id="pcit0290" dnum="WO002090355A"><text>WO002/090355</text></patcit>, <patcit id="pcit0291" dnum="WO03023561A"><text>WO03/023561</text></patcit>, <patcit id="pcit0292" dnum="WO03032991A"><text>WO03/032991</text></patcit>, and <patcit id="pcit0293" dnum="WO03037847A"><text>WO03/037847</text></patcit>; PDE inhibitors (e.g. compounds which slow the degradation of cyclic AMP (cAMP) and/or cyclic GMP (cGMP) by inhibition of the phosphodiesterases, which can lead to a relative increase in the intracellular concentration of cAMP and cGMP; possible PDE inhibitors are primarily those substances which are to be numbered among the class consisting of the PDE3 inhibitors, the class consisting of the PDE4 inhibitors and/or the class consisting of the PDE5 inhibitors, in particular those substances which can be designated as mixed types of PDE3/4 inhibitors or as mixed types of PDE3/4/5 inhibitors) such as those disclosed in patent publications <patcit id="pcit0294" dnum="DE1470341"><text>DE1470341</text></patcit>, <patcit id="pcit0295" dnum="DE2108438"><text>DE2108438</text></patcit>, <patcit id="pcit0296" dnum="DE2123328"><text>DE2123328</text></patcit>, <patcit id="pcit0297" dnum="DE2305339"><text>DE2305339</text></patcit>, <patcit id="pcit0298" dnum="DE2305575"><text>DE2305575</text></patcit>, <patcit id="pcit0299" dnum="DE2315801"><text>DE2315801</text></patcit>, <patcit id="pcit0300" dnum="DE2402908"><text>DE2402908</text></patcit>, <patcit id="pcit0301" dnum="DE2413935"><text>DE2413935</text></patcit>, <patcit id="pcit0302" dnum="DE2451417"><text>DE2451417</text></patcit>, <patcit id="pcit0303" dnum="DE2459090"><text>DE2459090</text></patcit>, <patcit id="pcit0304" dnum="DE2646469"><text>DE2646469</text></patcit>, <patcit id="pcit0305" dnum="DE2727481"><text>DE2727481</text></patcit>, <patcit id="pcit0306" dnum="DE2825048"><text>DE2825048</text></patcit>, <patcit id="pcit0307" dnum="DE2837161"><text>DE2837161</text></patcit>, <patcit id="pcit0308" dnum="DE2845220"><text>DE2845220</text></patcit>, <patcit id="pcit0309" dnum="DE2847621"><text>DE2847621</text></patcit>, <patcit id="pcit0310" dnum="DE2934747"><text>DE2934747</text></patcit>, <patcit id="pcit0311" dnum="DE3021792"><text>DE3021792</text></patcit>, <patcit id="pcit0312" dnum="DE3038166"><text>DE3038166</text></patcit>, <patcit id="pcit0313" dnum="DE3044568"><text>DE3044568</text></patcit>, <patcit id="pcit0314" dnum="EP000718A"><text>EP000718</text></patcit>, <patcit id="pcit0315" dnum="EP0008408A"><text>EP0008408</text></patcit>, <patcit id="pcit0316" dnum="EP0010759A"><text>EP0010759</text></patcit>, <patcit id="pcit0317" dnum="EP0059948A"><text>EP0059948</text></patcit>, <patcit id="pcit0318" dnum="EP0075436A"><text>EP0075436</text></patcit>, <patcit id="pcit0319" dnum="EP0096517A"><text>EP0096517</text></patcit>, <patcit id="pcit0320" dnum="EPO112987A"><text>EPO1 12987</text></patcit>, <patcit id="pcit0321" dnum="EPO116948A"><text>EPO1 16948</text></patcit>, <patcit id="pcit0322" dnum="EP0150937A"><text>EP0150937</text></patcit>, <patcit id="pcit0323" dnum="EP0158380A"><text>EP0158380</text></patcit>, <patcit id="pcit0324" dnum="EP0161632A"><text>EP0161632</text></patcit>, <patcit id="pcit0325" dnum="EP0161918A"><text>EP0161918</text></patcit>, <patcit id="pcit0326" dnum="EP0167121A"><text>EP0167121</text></patcit>, <patcit id="pcit0327" dnum="EP0199127A"><text>EP0199127</text></patcit>, <patcit id="pcit0328" dnum="EP0220044A"><text>EP0220044</text></patcit>, <patcit id="pcit0329" dnum="EP0247725A"><text>EP0247725</text></patcit>, <patcit id="pcit0330" dnum="EP0258191A"><text>EP0258191</text></patcit>, <patcit id="pcit0331" dnum="EP0272910A"><text>EP0272910</text></patcit>, <patcit id="pcit0332" dnum="EP0272914A"><text>EP0272914</text></patcit>, <patcit id="pcit0333" dnum="EP0294647A"><text>EP0294647</text></patcit>, <patcit id="pcit0334" dnum="EP0300726A"><text>EP0300726</text></patcit>, <patcit id="pcit0335" dnum="EP0335386A"><text>EP0335386</text></patcit>, <patcit id="pcit0336" dnum="EP0357788A"><text>EP0357788</text></patcit>, <patcit id="pcit0337" dnum="EP0389282A"><text>EP0389282</text></patcit>, <patcit id="pcit0338" dnum="EP0406958A"><text>EP0406958</text></patcit>, <patcit id="pcit0339" dnum="EP0426180A"><text>EP0426180</text></patcit>, <patcit id="pcit0340" dnum="EP0428302A"><text>EP0428302</text></patcit>, <patcit id="pcit0341" dnum="EP0435811A"><text>EP043581 1</text></patcit>, <patcit id="pcit0342" dnum="EP0470805A"><text>EP0470805</text></patcit>, <patcit id="pcit0343" dnum="EP0482208A"><text>EP0482208</text></patcit>, <patcit id="pcit0344" dnum="EP0490823A"><text>EP0490823</text></patcit>, <patcit id="pcit0345" dnum="EP0506194A"><text>EP0506194</text></patcit>, <patcit id="pcit0346" dnum="EP0511865A"><text>EP0511865</text></patcit>, <patcit id="pcit0347" dnum="EP0527117A"><text>EP0527117</text></patcit>, <patcit id="pcit0348" dnum="EP0626939A"><text>EP0626939</text></patcit>, <patcit id="pcit0349" dnum="EP0664289A"><text>EP0664289</text></patcit>, <patcit id="pcit0350" dnum="EP0671389A"><text>EP0671389</text></patcit>, <patcit id="pcit0351" dnum="EP0685474A"><text>EP0685474</text></patcit>, <patcit id="pcit0352" dnum="EP0685475A"><text>EP0685475</text></patcit>, <patcit id="pcit0353" dnum="EP0685479A"><text>EP0685479</text></patcit>, <patcit id="pcit0354" dnum="JP92234389B"><text>JP92234389</text></patcit>, <patcit id="pcit0355" dnum="JP94329652B"><text>JP94329652</text></patcit>, <patcit id="pcit0356" dnum="JP95010875B"><text>JP95010875</text></patcit>, <patcit id="pcit0357" dnum="US4963561A"><text>US4963561</text></patcit>, <patcit id="pcit0358" dnum="US5141931A"><text>US5141931</text></patcit>, <patcit id="pcit0359" dnum="WO9117991A"><text>WO9117991</text></patcit>, <patcit id="pcit0360" dnum="WO9200968A"><text>WO9200968</text></patcit>, <patcit id="pcit0361" dnum="WO9212961A"><text>WO9212961</text></patcit>, <patcit id="pcit0362" dnum="WO9307146A"><text>WO9307146</text></patcit>, <patcit id="pcit0363" dnum="WO9315044A"><text>WO9315044</text></patcit>, <patcit id="pcit0364" dnum="WO9315045A"><text>WO9315045</text></patcit>, <patcit id="pcit0365" dnum="WO9318024A"><text>WO9318024</text></patcit>, <patcit id="pcit0366" dnum="WO9319068A"><text>WO9319068</text></patcit>, <patcit id="pcit0367" dnum="WO9319720A"><text>WO9319720</text></patcit>, <patcit id="pcit0368" dnum="WO9319747A"><text>WO9319747</text></patcit>, <patcit id="pcit0369" dnum="WO9319749A"><text>WO9319749</text></patcit>, <patcit id="pcit0370" dnum="WO9319751A"><text>WO9319751</text></patcit>, <patcit id="pcit0371" dnum="WO9325517A"><text>WO9325517</text></patcit>, <patcit id="pcit0372" dnum="WO9402465A"><text>WO9402465</text></patcit>, <patcit id="pcit0373" dnum="WO9406423A"><text>WO9406423</text></patcit>, <patcit id="pcit0374" dnum="WO9412461A"><text>WO9412461</text></patcit>, <patcit id="pcit0375" dnum="WO9420455A"><text>WO9420455</text></patcit>, <patcit id="pcit0376" dnum="WO9422852A"><text>WO9422852</text></patcit>, <patcit id="pcit0377" dnum="WO9425437A"><text>WO9425437</text></patcit>, <patcit id="pcit0378" dnum="WO9427947A"><text>WO9427947</text></patcit>, <patcit id="pcit0379" dnum="WO9500516A"><text>WO9500516</text></patcit>, <patcit id="pcit0380" dnum="WO9501980A"><text>WO9501980</text></patcit>, <patcit id="pcit0381" dnum="WO9503794A"><text>WO9503794</text></patcit>, <patcit id="pcit0382" dnum="WO9504045A"><text>WO9504045</text></patcit>, <patcit id="pcit0383" dnum="WO9504046A"><text>WO9504046</text></patcit>, <patcit id="pcit0384" dnum="WO9505386A"><text>WO9505386</text></patcit>, <patcit id="pcit0385" dnum="WO9508534A"><text>WO9508534</text></patcit>, <patcit id="pcit0386" dnum="WO9509623A"><text>WO9509623</text></patcit>, <patcit id="pcit0387" dnum="WO9509624A"><text>WO9509624</text></patcit>, <patcit id="pcit0388" dnum="WO9509627A"><text>WO9509627</text></patcit>, <patcit id="pcit0389" dnum="WO9509836A"><text>WO9509836</text></patcit>, <patcit id="pcit0390" dnum="WO9514667A"><text>WO9514667</text></patcit>, <patcit id="pcit0391" dnum="WO9514680A"><text>WO9514680</text></patcit>, <patcit id="pcit0392" dnum="WO9514681A"><text>WO9514681</text></patcit>, <patcit id="pcit0393" dnum="WO9517392A"><text>WO9517392</text></patcit>, <patcit id="pcit0394" dnum="WO9517399A"><text>WO9517399</text></patcit>, <patcit id="pcit0395" dnum="WO9519362A"><text>WO9519362</text></patcit>, <patcit id="pcit0396" dnum="WO9522520A"><text>WO9522520</text></patcit>, <patcit id="pcit0397" dnum="WO9524381A"><text>WO9524381</text></patcit>, <patcit id="pcit0398" dnum="WO9527692A"><text>WO9527692</text></patcit>, <patcit id="pcit0399" dnum="WO9528926A"><text>WO9528926</text></patcit>, <patcit id="pcit0400" dnum="WO9535281A"><text>WO9535281</text></patcit>, <patcit id="pcit0401" dnum="WO9535282A"><text>WO9535282</text></patcit>, <patcit id="pcit0402" dnum="WO9600218A"><text>WO9600218</text></patcit>, <patcit id="pcit0403" dnum="WO9601825A"><text>WO9601825</text></patcit>, <patcit id="pcit0404" dnum="WO9602541A"><text>WO9602541</text></patcit>, <patcit id="pcit0405" dnum="WO9611917A"><text>WO9611917</text></patcit>, <patcit id="pcit0406" dnum="DE3142982"><text>DE3142982</text></patcit>, <patcit id="pcit0407" dnum="DEL116676"><text>DEl 116676</text></patcit>, <patcit id="pcit0408" dnum="DE2162096"><text>DE2162096</text></patcit>, <patcit id="pcit0409" dnum="EP0293063A"><text>EP0293063</text></patcit>, <patcit id="pcit0410" dnum="EP0463756A"><text>EP0463756</text></patcit>, <patcit id="pcit0411" dnum="EP0482208A"><text>EP0482208</text></patcit>, <patcit id="pcit0412" dnum="EP0579496A"><text>EP0579496</text></patcit>, <patcit id="pcit0413" dnum="EP0667345A"><text>EP0667345</text></patcit><patcit id="pcit0414" dnum="US6331543B"><text>US6331543</text></patcit>, <patcit id="pcit0415" dnum="US20050004222A"><text>US20050004222</text></patcit> (including those disclosed in formulas I- XIII and paragraphs 37-39, 85-0545 and 557-577), <patcit id="pcit0416" dnum="WO9307124A"><text>WO9307124</text></patcit>, <patcit id="pcit0417" dnum="EP0163965A"><text>EP0163965</text></patcit>, <patcit id="pcit0418" dnum="EP0393500A"><text>EP0393500</text></patcit>, <patcit id="pcit0419" dnum="EP0510562A"><text>EP0510562</text></patcit>, <patcit id="pcit0420" dnum="EP0553174A"><text>EP0553174</text></patcit>, <patcit id="pcit0421" dnum="WO9501338A"><text>WO9501338</text></patcit> and <patcit id="pcit0422" dnum="WO9603399A"><text>WO9603399</text></patcit>, as well as PDE5 inhibitors (such as RX-RA-69, SCH-51866, KT-734, vesnarinone, zaprinast, SKF-96231, ER-21355, BF/GP-385, NM-702 and sildenafil (Viagra^™)), PDE4 inhibitors (such as etazolate, ICI63197, RP73401, imazolidinone (RO-20-1724), MEM 1414 (R1533/R1500; Pharmacia Roche), denbufylline, rolipram, oxagrelate, nitraquazone, Y-590, DH-6471, SKF-94120, motapizone, lixazinone, indolidan, olprinone, atizoram, KS-506-G, dipanifylline, BMY-43351, atizoram, arofylline, filaminast, PDB-093, UCB-29646, CDP-840, SKF-107806, piclamilast, RS-17597, RS-25344- 000, SB-207499, TIBENELAST, SB-210667, SB-211572, SB-211600, SB-212066, SB-212179, GW-3600, CDP-840, mopidamol, anagrelide, ibudilast, amrinone, pimobendan, cilostazol, quazinone and N-(3,5-dichloropyrid-4-yl)-3-cyclopropylmethoxy4-difluoromethoxybenzamide, PDE3 inhibitors (such as ICI153, 100, bemorandane (RWJ 22867), MCI-154, UD-CG 212, sulmazole, ampizone, cilostainide, carbazeran, piroximone, imazodan, CL930, siguazodan, adibendan, saterinone, SKF-95654, SDZ-MKS-492, 349-U-85, emoradan, EMD-53998, EMD- 57033, NSP-306, NSP-307, revizinone, NM-702, WIN-62582 and WIN-63291, enoximone and milrinone, PDE3/4 inhibitors (such as benafentrine, trequinsin, ORO-30029, zardaverine, L- 686398, SDZ-ISQ-844, ORG-20241, EMD-54622, and tolafentrine) and other PDE inhibitors (such as vinpocetin, papaverine, enprofylline, cilomilast, fenoximone, pentoxifylline, roflumilast, tadalafil(Cialis^®), theophylline, and vardenafil(Levitra^®); Neuropeptide Y2 (NPY2) agonists include but are not limited to: polypeptide YY and fragments and variants thereof (e.g. YY3-36 (PYY3-36 )(N. Engl. J. Med. 349:941, 2003; IKPEAPGE DASPEELNRY YASLRHYLNL VTRQRY (SEQ ID NO:XXX)) and PYY agonists such as those disclosed in <patcit id="pcit0423" dnum="WO0247712A"><text>WO02/47712</text></patcit>, <patcit id="pcit0424" dnum="WO03026591A"><text>WO03/026591</text></patcit>, <patcit id="pcit0425" dnum="WO03057235A"><text>WO03/057235</text></patcit>, and <patcit id="pcit0426" dnum="WO03027637A"><text>WO03/027637</text></patcit>; serotonin reuptake inhibitors, such as, paroxetine, fluoxetine (Prozac^™), fluvoxamine, sertraline, citalopram, and imipramine, and those disclosed in <patcit id="pcit0427" dnum="US6162805A"><text>US6162805</text></patcit>, <patcit id="pcit0428" dnum="US6365633B"><text>US6365633</text></patcit>, <patcit id="pcit0429" dnum="WO0300663A"><text>WO03/00663</text></patcit>, <patcit id="pcit0430" dnum="WOO127060A"><text>WOO 1/27060</text></patcit>, and <patcit id="pcit0431" dnum="WOO1162341A"><text>WOO 1/162341</text></patcit>; thyroid hormone β agonists, such as KB-2611 (KaroBioBMS), and those disclosed in <patcit id="pcit0432" dnum="WO0215845A"><text>WO02/15845</text></patcit>, <patcit id="pcit0433" dnum="WO9721993A"><text>WO97/21993</text></patcit>, <patcit id="pcit0434" dnum="WO9900353A"><text>WO99/00353</text></patcit>, <patcit id="pcit0435" dnum="GB98284425A"><text>GB98/284425</text></patcit>, <patcit id="pcit0436" dnum="US183223"><text>U.S. Provisional Application No. 60/183,223</text></patcit>, and <patcit id="pcit0437" dnum="JP2000256190B"><text>Japanese Patent Application No. JP 2000256190</text></patcit>; 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-napthalenyl)-1-propenyl]benzoic acid (TTNPB), retinoic acid, and those disclosed in <patcit id="pcit0438" dnum="WO9900123A"><text>WO99/00123</text></patcit>; 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, Trecadrine, Zeneca D7114, N-5984 (Nisshin Kyorin), LY-377604 (Lilly), SR 59119A, and those disclosed in <patcit id="pcit0439" dnum="US5541204A"><text>US5541204</text></patcit>, <patcit id="pcit0440" dnum="US5770615A"><text>US5770615</text></patcit>, <patcit id="pcit0441" dnum="US5491134A"><text>US5491134</text></patcit>, <patcit id="pcit0442" dnum="US5776983A"><text>US5776983</text></patcit>, <patcit id="pcit0443" dnum="US488064A"><text>US488064</text></patcit>, <patcit id="pcit0444" dnum="US5705515A"><text>US5705515</text></patcit>, <patcit id="pcit0445" dnum="US5451677A"><text>US5451677</text></patcit>, <patcit id="pcit0446" dnum="WO9418161A"><text>WO94/18161</text></patcit>, <patcit id="pcit0447" dnum="WO9529159A"><text>WO95/29159</text></patcit>, <patcit id="pcit0448" dnum="WO9746556A"><text>WO97/46556</text></patcit>, <patcit id="pcit0449" dnum="WO9804526A"><text>WO98/04526</text></patcit> and <patcit id="pcit0450" dnum="WO9832753A"><text>WO98/32753</text></patcit>, <patcit id="pcit0451" dnum="WO0174782A"><text>WO01/74782</text></patcit>, <patcit id="pcit0452" dnum="WO0232897A"><text>WO02/32897</text></patcit>, <patcit id="pcit0453" dnum="WO03014113A"><text>WO03/014113</text></patcit>, <patcit id="pcit0454" dnum="WO03016276A"><text>WO03/016276</text></patcit>, <patcit id="pcit0455" dnum="WO03016307A"><text>WO03/016307</text></patcit>, <patcit id="pcit0456" dnum="WO03024948A"><text>WO03/024948</text></patcit>, <patcit id="pcit0457" dnum="WO03024953A"><text>WO03/024953</text></patcit> and <patcit id="pcit0458" dnum="WO03037881A"><text>WO03/037881</text></patcit>; noradrenergic agents 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, Oxydess II, Robese, 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 Benzenemethanol, alpha-(1-aminoethyl)-, hydrochloride), phentermine ((or Phenol, 3-[[4,5-duhydro-1H-imidazol-2-yl)ethyl](4-methylpheny-1)amino], monohydrochloride) such as Adipex-P^®, Lemmon, FASTING, Smith-Kline Beecham and Ionamin^®, Medeva), phendimetrazine ((or (2S,3S)-3,4-Dimethyl-2phenylmorpholine L-(+)- tartrate (1 :1)) such as Metra^® (Forest) , Plegine^® (Wyeth- Ay erst), Prelu-2^® (Boehringer Ingelheim), and Statobex^® (Lemmon), phendamine tartrate (such as Thephorin^® (2,3,4,9- Tetrahydro-2-methyl-9-phenyl-1H-indenol[2,1-c]pyridine L-(+)-tartrate (1 :1)), Hoffmann- LaRoche), methamphetamine (such as Desoxyn^®, Abbot ((S)-N, (alpha)-dimethylbenzeneethanamine hydrochloride)), and phendimetrazine tartrate (such as Bontril^® Slow-Release Capsules, Amarin (-3,4-Dimethyl-2-phenylmorpholine Tartrate); fatty acid oxidation upregulator/inducers such as Famoxin^® (Genset), monamine oxidase inhibitors including but not limited to befloxatone, moclobemide, brofaromine, phenoxathine, esuprone, befol, toloxatone, pirlindol, amiflamine, sercloremine, bazinaprine, lazabemide, milacemide, caroxazone and other certain compounds as disclosed by <patcit id="pcit0459" dnum="WO0112176A"><text>WO01/12176</text></patcit>; and other anti-obesity agents such as 5HT-2 agonists, ACC (acetyl-CoA carboxylase) inhibitors such as those described in <patcit id="pcit0460" dnum="WO03072197A"><text>WO03/072197</text></patcit>, alpha-lipoic acid (alpha-LA), AOD9604, appetite suppressants such as those in <patcit id="pcit0461" dnum="WO0340107A"><text>WO03/40107</text></patcit>, ATL-962 (Alizyme PLC), benzocaine, benzphetamine hydrochloride (Didrex), bladderwrack (focus vesiculosus), BRS3 (bombesin receptor subtype 3) agonists, bupropion, caffeine, CCK agonists, chitosan, chromium, conjugated linoleic acid, corticotropin-releasing hormone agonists, dehydroepiandrosterone, DGATl (diacylglycerol acyltransferase 1) inhibitors, DGA T2 (diacylglycerol acyltransferase 2) inhibitors, dicarboxylate transporter inhibitors, ephedra, exendin-4 (an inhibitor of glp-1) FAS (fatty acid synthase) inhibitors (such as Cerulenin and C75), fat resorption inhibitors (such as those in <patcit id="pcit0462" dnum="WO03053451A"><text>WO03/053451</text></patcit>, and the like), fatty acid transporter inhibitors, natural water soluble fibers (such as psyllium, plantago, guar, oat, pectin), galanin antagonists, galega (Goat's Rue, French Lilac), garcinia cambogia, germander (teucrium chamaedrys), ghrelin antibodies and ghrelin antagonists (such as those disclosed in <patcit id="pcit0463" dnum="WO0187335A"><text>WO01/87335</text></patcit>, and <patcit id="pcit0464" dnum="WO0208250A"><text>WO02/08250</text></patcit>), polypeptide hormones and variants thereof which affect the islet cell secretion, such as the hormones of the secretin/gastric inhibitory polypeptide (GIP)/vasoactive intestinal polypeptide (VIP)/pituitary adenylate cyclase activating polypeptide (PACAP)/glucagon-like polypeptide II (GLP- II)/glicentin/glucagon gene family and/or those of the adrenomedullin/amylin/calcitonin gene related polypeptide (CGRP) gene family includingGLP-1 (glucagon- like polypeptide 1) agonists (e.g. (1) exendin-4, (2) those GLP-I molecules described in <patcit id="pcit0465" dnum="US20050130891A"><text>US20050130891</text></patcit> including GLP- 1(7-34), GLP-1(7-35), GLP-1(7-36) or GLP-1(7-37) in its C-terminally carboxylated or amidated form or as modified GLP-I polypeptides and modifications thereof including those described in paragraphs 17-44 of <patcit id="pcit0466" dnum="US20050130891A"><text>US20050130891</text></patcit>, and derivatives derived from GLP-1-(7- 34)COOH and the corresponding acid amide are employed which have the following general formula: R-NH-HAEGTFTSDVSYLEGQAAKEFIAWLVK-CONH₂ wherein R=H or an organic compound having from 1 to 10 carbon atoms. Preferably, R is the residue of a carboxylic acid. 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 those disclosed and specifically described in <patcit id="pcit0467" dnum="US5536716A"><text>US5536716</text></patcit>), interleukin-6 (IL-6) and modulators thereof (as in <patcit id="pcit0468" dnum="WO03057237A"><text>WO03/057237</text></patcit>, and the like), L- carnitine, Mc3r (melanocortin 3 receptor) agonists, MCH2R (melanin concentrating hormone 2R) agonist/antagonists, melanin concentrating hormone antagonists, melanocortin agonists (such as Melanotan II or those described in <patcit id="pcit0469" dnum="WO9964002A"><text>WO 99/64002</text></patcit> and <patcit id="pcit0470" dnum="WO0074679A"><text>WO 00/74679</text></patcit>), nomame herba, phosphate transporter inhibitors, phytopharm compound 57 (CP 644,673), pyruvate, SCD-I (stearoyl-CoA_ desaturase-1) inhibitors, T71 (Tularik, Inc., Boulder CO), Topiramate (Topimax^®, indicated as an anti-convulsant which has been shown to increase weight loss), transcription factor modulators (such as those disclosed in <patcit id="pcit0471" dnum="WO03026576A"><text>WO03/026576</text></patcit>), B-hydroxy steroid dehydrogenase- 1 inhibitors (β -HSD-I), β-hydroxy-β-methylbutyrate, p57 (Pfizer), Zonisamide (Zonegran^™, indicated as an anti-epileptic which has been shown to lead to weight loss), and the agents disclosed in <patcit id="pcit0472" dnum="US20030119428A"><text>US20030119428</text></patcit> paragraphs 20-26.

1.2.2.7

Phosphodiesterase inhibitors

0198In certain embodiments, the regimen of combination therapy includes the administration of one or more phosphodiesterase ("PDE") inhibitors. PDE inhibitors slow the degradation of cyclic AMP (cAMP) and/or cyclic GMP (cGMP) by inhibiting 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 those substances which can be designated as mixed types of PDE3/4 inhibitors or as mixed types of PDE3/4/5 inhibitors. Non-limiting examples of such PDE inhibitors are described in the following patent applications and patents: <patcit id="pcit0473" dnum="DE1470341"><text>DE1470341</text></patcit>, <patcit id="pcit0474" dnum="DE2108438"><text>DE2108438</text></patcit>, <patcit id="pcit0475" dnum="DE2123328"><text>DE2123328</text></patcit>, <patcit id="pcit0476" dnum="DE2305339"><text>DE2305339</text></patcit>, <patcit id="pcit0477" dnum="DE2305575"><text>DE2305575</text></patcit>, <patcit id="pcit0478" dnum="DE2315801"><text>DE2315801</text></patcit>, <patcit id="pcit0479" dnum="DE2402908"><text>DE2402908</text></patcit>, <patcit id="pcit0480" dnum="DE2413935"><text>DE2413935</text></patcit>, <patcit id="pcit0481" dnum="DE2451417"><text>DE2451417</text></patcit>, <patcit id="pcit0482" dnum="DE2459090"><text>DE2459090</text></patcit>, <patcit id="pcit0483" dnum="DE2646469"><text>DE2646469</text></patcit>, <patcit id="pcit0484" dnum="DE2727481"><text>DE2727481</text></patcit>, <patcit id="pcit0485" dnum="DE2825048"><text>DE2825048</text></patcit>, <patcit id="pcit0486" dnum="DE2837161"><text>DE2837161</text></patcit>, <patcit id="pcit0487" dnum="DE2845220"><text>DE2845220</text></patcit>, <patcit id="pcit0488" dnum="DE2847621"><text>DE2847621</text></patcit>, <patcit id="pcit0489" dnum="DE2934747"><text>DE2934747</text></patcit>, <patcit id="pcit0490" dnum="DE3021792"><text>DE3021792</text></patcit>, <patcit id="pcit0491" dnum="DE3038166"><text>DE3038166</text></patcit>, <patcit id="pcit0492" dnum="DE3044568"><text>DE3044568</text></patcit>, <patcit id="pcit0493" dnum="EP000718A"><text>EP000718</text></patcit>, <patcit id="pcit0494" dnum="EP0008408A"><text>EP0008408</text></patcit>, <patcit id="pcit0495" dnum="EP0010759A"><text>EP0010759</text></patcit>, <patcit id="pcit0496" dnum="EP0059948A"><text>EP0059948</text></patcit>, <patcit id="pcit0497" dnum="EP0075436A"><text>EP0075436</text></patcit>, <patcit id="pcit0498" dnum="EP0096517A"><text>EP0096517</text></patcit>, <patcit id="pcit0499" dnum="EPO112987A"><text>EPO1 12987</text></patcit>, <patcit id="pcit0500" dnum="EPO116948A"><text>EPO1 16948</text></patcit>, <patcit id="pcit0501" dnum="EP0150937A"><text>EP0150937</text></patcit>, <patcit id="pcit0502" dnum="EP0158380A"><text>EP0158380</text></patcit>, <patcit id="pcit0503" dnum="EP0161632A"><text>EP0161632</text></patcit>, <patcit id="pcit0504" dnum="EP0161918A"><text>EP0161918</text></patcit>, <patcit id="pcit0505" dnum="EP0167121A"><text>EP0167121</text></patcit>, <patcit id="pcit0506" dnum="EP0199127A"><text>EP0199127</text></patcit>, <patcit id="pcit0507" dnum="EP0220044A"><text>EP0220044</text></patcit>, <patcit id="pcit0508" dnum="EP0247725A"><text>EP0247725</text></patcit>, <patcit id="pcit0509" dnum="EP0258191A"><text>EP0258191</text></patcit>, <patcit id="pcit0510" dnum="EP0272910A"><text>EP0272910</text></patcit>, <patcit id="pcit0511" dnum="EP0272914A"><text>EP0272914</text></patcit>, <patcit id="pcit0512" dnum="EP0294647A"><text>EP0294647</text></patcit>, <patcit id="pcit0513" dnum="EP0300726A"><text>EP0300726</text></patcit>, <patcit id="pcit0514" dnum="EP0335386A"><text>EP0335386</text></patcit>, <patcit id="pcit0515" dnum="EP0357788A"><text>EP0357788</text></patcit>, <patcit id="pcit0516" dnum="EP0389282A"><text>EP0389282</text></patcit>, <patcit id="pcit0517" dnum="EP0406958A"><text>EP0406958</text></patcit>, <patcit id="pcit0518" dnum="EP0426180A"><text>EP0426180</text></patcit>, <patcit id="pcit0519" dnum="EP0428302A"><text>EP0428302</text></patcit>, <patcit id="pcit0520" dnum="EP0435811A"><text>EP0435811</text></patcit>, <patcit id="pcit0521" dnum="EP0470805A"><text>EP0470805</text></patcit>, <patcit id="pcit0522" dnum="EP0482208A"><text>EP0482208</text></patcit>, <patcit id="pcit0523" dnum="EP0490823A"><text>EP0490823</text></patcit>, <patcit id="pcit0524" dnum="EP0506194A"><text>EP0506194</text></patcit>, <patcit id="pcit0525" dnum="EP0511865A"><text>EP0511865</text></patcit>, <patcit id="pcit0526" dnum="EP0527117A"><text>EP0527117</text></patcit>, <patcit id="pcit0527" dnum="EP0626939A"><text>EP0626939</text></patcit>, <patcit id="pcit0528" dnum="EP0664289A"><text>EP0664289</text></patcit>, <patcit id="pcit0529" dnum="EP0671389A"><text>EP0671389</text></patcit>, <patcit id="pcit0530" dnum="EP0685474A"><text>EP0685474</text></patcit>, <patcit id="pcit0531" dnum="EP0685475A"><text>EP0685475</text></patcit>, <patcit id="pcit0532" dnum="EP0685479A"><text>EP0685479</text></patcit>, <patcit id="pcit0533" dnum="JP92234389B"><text>JP92234389</text></patcit>, <patcit id="pcit0534" dnum="JP94329652B"><text>JP94329652</text></patcit>, <patcit id="pcit0535" dnum="JP95010875B"><text>JP95010875</text></patcit>, <patcit id="pcit0536" dnum="US4963561A"><text>U.S. Pat. Nos. 4,963,561</text></patcit>, <patcit id="pcit0537" dnum="US5141931A"><text>5,141,931</text></patcit>, <patcit id="pcit0538" dnum="WO9117991A"><text>WO9117991</text></patcit>, <patcit id="pcit0539" dnum="WO9200968A"><text>WO9200968</text></patcit>, <patcit id="pcit0540" dnum="WO9212961A"><text>WO9212961</text></patcit>, <patcit id="pcit0541" dnum="WO9307146A"><text>WO9307146</text></patcit>, <patcit id="pcit0542" dnum="WO9315044A"><text>WO9315044</text></patcit>, <patcit id="pcit0543" dnum="WO9315045A"><text>WO9315045</text></patcit>, <patcit id="pcit0544" dnum="WO9318024A"><text>WO9318024</text></patcit>, <patcit id="pcit0545" dnum="WO9319068A"><text>WO9319068</text></patcit>, <patcit id="pcit0546" dnum="WO9319720A"><text>WO9319720</text></patcit>, <patcit id="pcit0547" dnum="WO9319747A"><text>WO9319747</text></patcit>, <patcit id="pcit0548" dnum="WO9319749A"><text>WO9319749</text></patcit>, <patcit id="pcit0549" dnum="WO9319751A"><text>WO9319751</text></patcit>, <patcit id="pcit0550" dnum="WO9325517A"><text>WO9325517</text></patcit>, <patcit id="pcit0551" dnum="WO9402465A"><text>WO9402465</text></patcit>, <patcit id="pcit0552" dnum="WO9406423A"><text>WO9406423</text></patcit>, <patcit id="pcit0553" dnum="WO9412461A"><text>WO9412461</text></patcit>, <patcit id="pcit0554" dnum="WO9420455A"><text>WO9420455</text></patcit>, <patcit id="pcit0555" dnum="WO9422852A"><text>WO9422852</text></patcit>, <patcit id="pcit0556" dnum="WO9425437A"><text>WO9425437</text></patcit>, <patcit id="pcit0557" dnum="WO9427947A"><text>WO9427947</text></patcit>, <patcit id="pcit0558" dnum="WO9500516A"><text>WO9500516</text></patcit>, <patcit id="pcit0559" dnum="WO9501980A"><text>WO9501980</text></patcit>, <patcit id="pcit0560" dnum="WO09503794A"><text>WO09503794</text></patcit>, <patcit id="pcit0561" dnum="WO9504045A"><text>WO9504045</text></patcit>, <patcit id="pcit0562" dnum="WO9504046A"><text>WO9504046</text></patcit>, <patcit id="pcit0563" dnum="WO9505386A"><text>WO9505386</text></patcit>, <patcit id="pcit0564" dnum="WO9508534A"><text>WO9508534</text></patcit>, <patcit id="pcit0565" dnum="WO9509623A"><text>WO9509623</text></patcit>, <patcit id="pcit0566" dnum="WO9509624A"><text>WO9509624</text></patcit>, <patcit id="pcit0567" dnum="WO9509627A"><text>WO9509627</text></patcit>, <patcit id="pcit0568" dnum="WO9509836A"><text>WO9509836</text></patcit>, <patcit id="pcit0569" dnum="WO9514667A"><text>WO9514667</text></patcit>, <patcit id="pcit0570" dnum="WO9514680A"><text>WO9514680</text></patcit>, <patcit id="pcit0571" dnum="WO9514681A"><text>WO9514681</text></patcit>, <patcit id="pcit0572" dnum="WO9517392A"><text>WO9517392</text></patcit>, <patcit id="pcit0573" dnum="WO9517399A"><text>WO9517399</text></patcit>, <patcit id="pcit0574" dnum="WO9519362A"><text>WO9519362</text></patcit>, <patcit id="pcit0575" dnum="WO9522520A"><text>WO9522520</text></patcit>, <patcit id="pcit0576" dnum="WO9524381A"><text>WO9524381</text></patcit>, <patcit id="pcit0577" dnum="WO9527692A"><text>WO9527692</text></patcit>, <patcit id="pcit0578" dnum="WO9528926A"><text>WO9528926</text></patcit>, <patcit id="pcit0579" dnum="WO9535281A"><text>WO9535281</text></patcit>, <patcit id="pcit0580" dnum="WO9535282A"><text>WO9535282</text></patcit>, <patcit id="pcit0581" dnum="WO9600218A"><text>WO9600218</text></patcit>, <patcit id="pcit0582" dnum="WO9601825A"><text>WO9601825</text></patcit>, <patcit id="pcit0583" dnum="WO9602541A"><text>WO9602541</text></patcit>, <patcit id="pcit0584" dnum="WO9611917A"><text>WO9611917</text></patcit>, <patcit id="pcit0585" dnum="DE3142982"><text>DE3142982</text></patcit>, <patcit id="pcit0586" dnum="DE1116676"><text>DE1 116676</text></patcit>, <patcit id="pcit0587" dnum="DE2162096"><text>DE2162096</text></patcit>, <patcit id="pcit0588" dnum="EP0293063A"><text>EP0293063</text></patcit>, <patcit id="pcit0589" dnum="EP0463756A"><text>EP0463756</text></patcit>, <patcit id="pcit0590" dnum="EP0482208A"><text>EP0482208</text></patcit>, <patcit id="pcit0591" dnum="EP0579496A"><text>EP0579496</text></patcit>, <patcit id="pcit0592" dnum="EP0667345A"><text>EP0667345</text></patcit><patcit id="pcit0593" dnum="US6331543B"><text>US6,331,543</text></patcit>, <patcit id="pcit0594" dnum="US20050004222A"><text>US20050004222</text></patcit> (including those disclosed in formulas I-XIII and paragraphs 37-39, 85-0545 and 557-577) and <patcit id="pcit0595" dnum="WO9307124A"><text>WO9307124</text></patcit>, <patcit id="pcit0596" dnum="EP0163965A"><text>EP0163965</text></patcit>, <patcit id="pcit0597" dnum="EP0393500A"><text>EP0393500</text></patcit>, <patcit id="pcit0598" dnum="EP0510562A"><text>EP0510562</text></patcit>, <patcit id="pcit0599" dnum="EP0553174A"><text>EP0553174</text></patcit>, <patcit id="pcit0600" dnum="WO9501338A"><text>WO9501338</text></patcit> and <patcit id="pcit0601" dnum="WO9603399A"><text>WO9603399</text></patcit>. PDE5 inhibitors which may be mentioned by way of example are RX-RA-69, SCH-51866, KT-734, vesnarinone, zaprinast, SKF-96231, ER-21355, BF/GP-385, NM-702 and sildenafil (Viagra^®). PDE4 inhibitors which may be mentioned by way of example are RO-20-1724, MEM 1414 (R1533/R1500; Pharmacia Roche), DENBUFYLLINE, ROLIPRAM, OXAGRELATE, NITRAQUAZONE, Y-590, DH-6471, SKF-94120, MOTAPIZONE, LIXAZINONE, INDOLIDAN, OLPRINONE, ATIZORAM, KS-506-G, DIPAMFYLLINE, BMY-43351, ATIZORAM, AROFYLLINE, FILAMINAST, PDB-093, UCB-29646, CDP-840, SKF- 107806, PICLAMILAST, RS- 17597, RS-25344-000, SB-207499, TIBENELAST, SB-210667, SB-211572, SB-211600, SB-212066, SB-212179, GW-3600, CDP-840, MOPIDAMOL, ANAGRELIDE, IBUDILAST, AMRINONE, PIMOBENDAN, CILOSTAZOL, QUAZINONE and N-(3,5-dichloropyrid-4-yl)-3-cyclopropylmethoxy4-difluoromethoxybenzamide. PDE3 inhibitors which may be mentioned by way of example are SULMAZOLE, AMPIZONE, CILOSTAMIDE, CARBAZERAN, PIROXIMONE, IMAZODAN, CI-930, SIGUAZODAN-., ADIBENDAN, SATERINONE, SKF-95654, SDZ-MKS-492, 349-U-85, EMORADAN, EMD-53998, EMD-57033, NSP-306, NSP-307, REVIZINONE, NM-702, WIN-62582 and WIN-63291, ENOXIMONE and MILRINONE. PDE3/4 inhibitors which may be mentioned by way of example are BENAFENTRINE, TREQUINSIN, ORG-30029, ZARDAVERINE, L-686398, SDZ-ISQ-844, ORG-20241, EMD-54622, and TOLAFENTRINE. Other PDE inhibitors include: cilomilast, pentoxifylline, roflumilast, tadalafil(Cialis^®), theophylline, and vardenafil(Levitra^®), zaprinast (PDE5 specific). GCC AGONIST

1.2.2.8

Analgesic Agents

0199In certain embodiments, the regimen of combination therapy includes the administration of one or more analgesic agents, <i>e</i>.<i>g</i>., an analgesic compound or an analgesic polypeptide. In some embodiments, the GCC agonist formulation is administered simultaneously or sequentially with one or more analgesic agents. In other embodiments, the GCC agonist is covalently linked or attached to an analgesic agent to create a therapeutic conjugate. Non-limiting examples of analgesic agents that can be used include calcium channel blockers, 5HT receptor antagonists (for example 5HT3, 5HT4 and 5HT1 receptor antagonists), opioid receptor agonists (loperamide, fedotozine, and fentanyl), NK1 receptor antagonists, CCK receptor agonists (<i>e</i>.<i>g</i>., loxiglumide), NK1 receptor antagonists, NK3 receptor antagonists, norepinephrine-serotonin reuptake inhibitors (NSRI), vanilloid and cannabanoid receptor agonists, and sialorphin. Further examples of analgesic agents in the various classes are known in the art.

0200In one embodiment, the analgesic agent is an analgesic polypeptide selected from the group consisting of sialorphin-related polypeptides, including those comprising 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 to neprilysin and inhibit neprilysin-mediated breakdown of substance P and Met-enkephalin. Thus, compounds or polypeptides that are inhibitors of neprilysin are useful analgesic agents which can be administered with the GCC agonists described herein or covalently linked to a GCC agonist to form a therapeutic conjugate. Sialorphin and related polypeptides are described in <patcit id="pcit0602" dnum="US6589750B"><text>U.S. Patent 6,589,750</text></patcit>; <patcit id="pcit0603" dnum="US20030078200A1"><text>U.S. 20030078200 Al</text></patcit>; and <patcit id="pcit0604" dnum="WO02051435A2"><text>WO 02/051435 A2</text></patcit>.

0201In another embodiment, a GCC agonist formulation of the invention is administered as part of a regimen of combination therapy 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, methyl nalozone, nalmefene, cypridime, beta funaltrexamine, naloxonazine, naltrindole, nor-binaltorphimine, enkephalin pentapeptide (HOE825; Tyr-D-Lys-Gly-Phe-L-homoserine), trimebutine, vasoactive intestinal polypeptide, gastrin, glucagons. Non-limiting examples of opioid receptor agonists include fedotozine, asimadoline, and ketocyclazocine, the compounds described in <patcit id="pcit0605" dnum="WO03097051A"><text>WO03/097051</text></patcit> and <patcit id="pcit0606" dnum="WO05007626A"><text>WO05/007626</text></patcit>, morphine, diphenyloxylate, frakefamide (H-Tyr-D-Ala-Phe(F)-Phe-NH 2; <patcit id="pcit0607" dnum="WO01019849A1"><text>WO 01/019849 A1</text></patcit>), and loperamide.

0202Further non-limiting examples of analgesic agents that can be used in a regimen of combination therapy along with the GCC agonist formulations of the invention include the dipeptide Tyr-Arg (kyotorphin); the chromogranin-derived polypeptide (CgA _ 47-66; <i>See, e.g.,</i><nplcit id="ncit0032" npl-type="s"><text>Ghia et al. 2004 Regulatory polypeptides 119: 199</text></nplcit>); CCK receptor agonists such as caentlein; conotoxin polypeptides; peptide analogs of thymulin (<patcit id="pcit0608" dnum="FR2830451"><text>FR Application 2830451</text></patcit>); CCK (CCKa or CCKb) receptor antagonists, including loxiglumide and dexloxiglumide (the R- isomer of loxiglumide) (<patcit id="pcit0609" dnum="WO8805774A"><text>WO 88/05774</text></patcit>); 5-HT4 agonists such as tegaserod (Zelnorm^®), mosapride, metoclopramide, zacopride, cisapride, renzapride, benzimidazolone derivatives such as BIMU 1 and BIMU 8, and lirexapride; calcium channel blockers such as ziconotide and related compounds described in, for example, <patcit id="pcit0610" dnum="EP625162B1"><text>EP625162B1</text></patcit>, <patcit id="pcit0611" dnum="US5364842A"><text>US 5,364,842</text></patcit>, <patcit id="pcit0612" dnum="US5587454A"><text>US 5,587,454</text></patcit>, <patcit id="pcit0613" dnum="US5824645A"><text>US 5,824,645</text></patcit>, <patcit id="pcit0614" dnum="US5859186A"><text>US 5,859,186</text></patcit>, <patcit id="pcit0615" dnum="US5994305A"><text>US 5,994,305</text></patcit>, <patcit id="pcit0616" dnum="US6087091A"><text>US 6087,091</text></patcit>, <patcit id="pcit0617" dnum="US6136786A"><text>US 6,136,786</text></patcit>, <patcit id="pcit0618" dnum="WO9313128A1"><text>WO 93/13128 Al</text></patcit>, <patcit id="pcit0619" dnum="EP1336409A1"><text>EP 1336409 Al</text></patcit>, <patcit id="pcit0620" dnum="EP835126A1"><text>EP 835126 Al</text></patcit>, <patcit id="pcit0621" dnum="EP835126B1"><text>EP 835126 Bl</text></patcit>, <patcit id="pcit0622" dnum="US5795864A"><text>US 5,795,864</text></patcit>, <patcit id="pcit0623" dnum="US5891849A"><text>US 5,891,849</text></patcit>, <patcit id="pcit0624" dnum="US6054429A"><text>US 6,054,429</text></patcit>, <patcit id="pcit0625" dnum="WO9701351A1"><text>WO 97/01351 Al</text></patcit>; NK-I, receptor antagonists such as aprepitant (Merck &amp; Co Inc), vofopitant, ezlopitant (Pfizer, Inc.), R-673 (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, for example, <patcit id="pcit0626" dnum="EP873753A1"><text>EP 873753 Al</text></patcit>, <patcit id="pcit0627" dnum="US20010006972A1"><text>US 20010006972 Al</text></patcit>, <patcit id="pcit0628" dnum="US20030109417A1"><text>US 20030109417 Al</text></patcit>, <patcit id="pcit0629" dnum="WO0152844A1"><text>WO 01/52844 Al </text></patcit>(for a review see <nplcit id="ncit0033" npl-type="s"><text>Giardina et al. 2003.Drugs 6:758</text></nplcit>); NK-2 receptor antagonists such as nepadutant (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), SSR-241586, tahietant and related compounds described in, for example, <patcit id="pcit0630" dnum="WO02094187A2"><text>WO 02/094187 A2</text></patcit>, <patcit id="pcit0631" dnum="EP876347A1"><text>EP 876347 Al</text></patcit>, <patcit id="pcit0632" dnum="WO9721680A1"><text>WO 97/21680 Al</text></patcit>, <patcit id="pcit0633" dnum="US6277862B"><text>US 6,277,862</text></patcit>, <patcit id="pcit0634" dnum="WO9811090A"><text>WO 98/1 1090</text></patcit>, <patcit id="pcit0635" dnum="WO9528418A"><text>WO 95/28418</text></patcit>, <patcit id="pcit0636" dnum="WO9719927A"><text>WO 97/19927</text></patcit>, and <nplcit id="ncit0034" npl-type="s"><text>Boden et al. (J Med Chem. 39:1664-75, 1996</text></nplcit>); norepinephrine-serotonin reuptake inhibitors (NSRI) such as milnacipran and related compounds described in <patcit id="pcit0637" dnum="WO03077897A"><text>WO 03/077897</text></patcit>; and vanilloid receptor antagonists such as arvanil and related compouds described in <patcit id="pcit0638" dnum="WO0164212A1"><text>WO 01/64212 Al</text></patcit>.

0203In addition to sialorphin-related polypeptides, analgesic polypeptides include: AspPhe, endomorphin-1, endomorphin-2, nocistatin, dalargin, lupron, ziconotide, and substance P.

1.2.2.9 Insulin and Insulin Modulating Agents

0204The 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. Sources of human insulin include pharmaceutically acceptable and sterile formulations such as those available from Eli Lilly (Indianapolis, Ind. 46285) as Humulin^™ (human insulin rDNA origin). See, the <nplcit id="ncit0035" npl-type="b"><text>THE PHYSICIAN'S DESK REFERENCE, 55.sup.th Ed. (2001</text></nplcit>) Medical Economics, Thomson Healthcare (disclosing other suitable human insulins).

0205The GCC peptides described herein can also be used in combination therapy with agents that can boost insulin effects or levels of a subject upon administration, e.g. glipizide and/or rosiglitazone. The polypeptides and agonists described herein can be used in combitherapy with SYMLIN^® (pramlintide acetate) and Exenatide^® (synthetic exendin-4; a 39 aa polypeptide).

1.2.2.10

Anti-Hypertensive Agents

0206The GCC agonist peptides described herein can be used in combination therapy with an anti-hypertensive 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 agents, such as amiloride, and triamterene; carbonic anhydrase inhibitors, osmotics(such as glycerin) 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, penbutolol, pindolol, propanolol, sotalol, tertatolol, tilisolol, and timolol, and the like; (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, nimodepine, nisoldipine, nitrendipine, manidipine, pranidipine, and verapamil, and the like; (4) angiotensin converting enzyme (ACE) inhibitors such as benazepril; captopril; ceranapril; cilazapril; delapril; enalapril; enalopril; fosinopril; imidapril; lisinopril; losinopril; moexipril; quinapril; quinaprilat; ramipril; perindopril; peritidropril, quanipril; spirapril; tenocapril; trandolapril, and zofenopril, and the like; (5) neutral endopeptidase inhibitors such as omapatrilat, cadoxatril and ecadotril, fosidotril, sampatrilat, AVE7688, ER4030, and the like; (6) endothelin antagonists such as tezosentan, 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, and the like; (9) α/β adrenergic blockers such as nipradilol, arotinolol and amosulalol, and the like; (10) alpha 1 blockers, such as terazosin, urapidil, prazosin, tamsulosin, bunazosin, trimazosin, doxazosin, naftopidil, indoramin, WHP 164, and XENO1O, and the like; (11) alpha 2 agonists such as lofexidine, tiamenidine, inoxonidine, rilmenidine and guanobenz, and the like; (12) aldosterone inhibitors, and the like; and (13) angiopoietin-2 -binding agents such as those disclosed in <patcit id="pcit0639" dnum="WO03030833A"><text>WO03/030833</text></patcit>. Specific anti-hypertensive agents that can be used in combination with polypeptides and agonists described herein include, but are not limited to: diuretics, such as thiazides (e.g., chlorthalidone, cyclothiazide (<nplcit id="ncit0036" npl-type="c"><text>CAS RN 2259-96-3</text></nplcit>), chlorothiazide (<nplcit id="ncit0037" npl-type="c"><text>CAS RN 72956-09-3</text></nplcit>, which may be prepared as disclosed in <patcit id="pcit0640" dnum="US2809194A"><text>US2809194</text></patcit>), dichlorophenamide, hydroflumethiazide, indapamide, polythiazide, bendroflumethazide, methyclothazide, polythiazide, trichlormethazide, chlorthalidone, indapamide, metolazone, quinethazone, althiazide (<nplcit id="ncit0038" npl-type="c"><text>CAS RN 5588-16-9</text></nplcit>, which may be prepared as disclosed in <patcit id="pcit0641" dnum="GB902658A"><text>British Patent No. 902,658</text></patcit>), benzthiazide (<nplcit id="ncit0039" npl-type="c"><text>CAS RN 91-33-8</text></nplcit>, which may be prepared as disclosed in <patcit id="pcit0642" dnum="US3108097A"><text>US3108097</text></patcit>), buthiazide (which may be prepared as disclosed in <patcit id="pcit0643" dnum="GB861367A"><text>British Patent Nos. 861 ,367</text></patcit>), and hydrochlorothiazide), loop diuretics (e.g. bumetanide, ethacrynic acid, furosemide, and torasemide), potassium sparing agents (e.g. amiloride, and triamterene (<nplcit id="ncit0040" npl-type="c"><text>CAS Number 396-01-OP</text></nplcit>)) and aldosterone antagonists (e.g. spironolactone (<nplcit id="ncit0041" npl-type="c"><text>CAS Number 52-01-7</text></nplcit>), epirenone, and the like); β-adrenergic blockers such as Amiodarone (Cordarone, Pacerone), bunolol hydrochloride (<nplcit id="ncit0042" npl-type="c"><text>CAS RN 31969-05-8</text></nplcit>, Parke-Davis), acebutolol (±N-[3-Acetyl-4-[2-hydroxy-3-[1 methylethyl)amino]propoxy]phenyl]-butanamide, or (±)-3'-Acetyl-4'-[2-hydroxy -3-(isopropylamino) propoxy] butyranilide), acebutolol hydrochloride (e.g. Sectral^®, Wyeth-Ayerst), alprenolol hydrochloride (<nplcit id="ncit0043" npl-type="c"><text>CAS RN 13707-88-5</text></nplcit> see <patcit id="pcit0644" dnum="NL6605692"><text>Netherlands Patent Application No. 6,605,692</text></patcit>), atenolol (e.g. Tenormin^®, AstraZeneca), carteolol hydrochloride (e.g. Cartrol^® Filmtab^®, Abbott), Celiprolol hydrochloride (<nplcit id="ncit0044" npl-type="c"><text>CAS RN 57470-78-7</text></nplcit>, also see in <patcit id="pcit0645" dnum="US4034009A"><text>US4034009</text></patcit>), cetamolol hydrochloride (<nplcit id="ncit0045" npl-type="c"><text>CAS RN 77590-95-5</text></nplcit>, see also <patcit id="pcit0646" dnum="US4059622A"><text>US4059622</text></patcit>), labetalol hydrochloride (e.g. Normodyne^®, Schering), esmolol hydrochloride (e.g. Brevibloc^®, Baxter), levobetaxolol hydrochloride (e.g. Betaxon^™ Ophthalmic Suspension, Alcon), levobunolol hydrochloride (e.g. Betagan^® Liquifilm^®) with C CAP^® Compliance Cap, Allergan), nadolol (e.g. Nadolol, Mylan), practolol (<nplcit id="ncit0046" npl-type="c"><text>CAS RN 6673-35-4</text></nplcit>, see also <patcit id="pcit0647" dnum="US3408387A"><text>US3408387</text></patcit>), propranolol hydrochloride (<nplcit id="ncit0047" npl-type="c"><text>CAS RN 318-98-9</text></nplcit>), sotalol hydrochloride (e.g. Betapace AF^™,Berlex), timolol (2-Propanol,1-[(1,1-dimethylethyl)amino]-3-[[4-4(4-morpholinyl)-l,2,5-thiadiazol-3-yl]oxy]-, hemihydrate, (S)-, <nplcit id="ncit0048" npl-type="c"><text>CAS RN 91524-16-2</text></nplcit>), timolol maleate (S)-I -[1,1-dimethylethyl) amino]-3-[[4- (4-morpholinyl)-l,2,5-thiadiazol -3- yl] oxy]-2-propanol (Z)-2-butenedioate (1 :1) salt, <nplcit id="ncit0049" npl-type="c"><text>CAS RN 26921-17-5</text></nplcit>), bisoprolol (2-Propanol, l-[4-[[2-(1-methylethoxy)ethoxy]-methyl]phenoxyl]-3-[(1-meth- ylethyl)amino]-, (±), <nplcit id="ncit0050" npl-type="c"><text>CAS RN 66722-44-9</text></nplcit>), bisoprolol fumarate (such as (±)-1-[4-[[2-(1-Methylethoxy) ethoxy]methyl]phenoxy]-3-[(1-methylethyl)amino]-2-propanol (E) -2-butenedioate (2:1) (salt), e.g., Zebeta^™ , Lederle Consumer), nebivalol (2H-1-Benzopyran-2-methanol, αα'-[iminobis(methylene)]bis[6-fluoro-3,4-dihydro-, <nplcit id="ncit0051" npl-type="c"><text>CAS RN 99200-09-6</text></nplcit> see also <patcit id="pcit0648" dnum="US4654362A"><text>U.S. Pat. No. 4,654,362</text></patcit>), cicloprolol hydrochloride, such 2-Propanol, l-[4-[2-(cyclopropylmethoxy)ethoxy]phenoxy]-3-[1-methylethyl)amino]-, hydrochloride, A.A.S. RN 63686-79-3), dexpropranolol hydrochloride (2-Propano1,1-[l-methylethy)-amino]-3-(1-naphthalenyloxy)-hydrochloride (<nplcit id="ncit0052" npl-type="c"><text>CAS RN 13071-11-9</text></nplcit>), diacetolol hydrochloride (Acetamide, N-[3-acetyl-4-[2-hydroxy-3-[(1-methyl-ethyl)amino]propoxy] [phenyl]-, monohydrochloride <nplcit id="ncit0053" npl-type="c"><text>CAS RN 69796-04-9</text></nplcit>), dilevalol hydrochloride (Benzamide, 2-hydroxy-5-[l-hydroxy-2-[l-methyl-3-phenylpropyl)amino]ethyl]-, monohydrochloride, <nplcit id="ncit0054" npl-type="c"><text>CAS RN 75659-08-4</text></nplcit>), exaprolol hydrochloride (2-Propanol, 1 -(2-cyclohexylphenoxy)-3 - [( 1 -methylethyl)amino] -, hydrochloride <nplcit id="ncit0055" npl-type="c"><text>CAS RN 59333-90-3</text></nplcit>), flestolol sulfate (Benzoic acid, 2-fluro-,3-[[2-[aminocarbonyl)amino]- - dimethylethyl]amino]-2-hydeoxtpropyl ester, (+)- sulfate (1 :1) (salt), <nplcit id="ncit0056" npl-type="c"><text>CAS RN 88844-73-9</text></nplcit>; metalol hydrochloride (Methanesulfonamide, -N-[4-[l-hydroxy-2-(methylamino)propyl]phenyl]-, monohydrochloride <nplcit id="ncit0057" npl-type="c"><text>CAS RN 1701-65-7</text></nplcit>), metoprolol 2-Propanol, l-[4-(2- methoxyethyl)phenoxy]-3-[l-methylethyl)amino]-; <nplcit id="ncit0058" npl-type="c"><text>CAS RN 37350-58-6</text></nplcit>), metoprolol tartrate (such as 2-Propanol, l-[4-(2-methoxyethyl)phenoxy]-3-[(1-methylethyl)amino]-, e.g., Lopressor^®, Novartis), pamatolol sulfate (Carbamic acid, [2-[4-[2-hydroxy-3-[(1- methylethyl)amino]propoxyl]phenyl]-ethyl]-, methyl ester, (±) sulfate (salt) (2:1), <nplcit id="ncit0059" npl-type="c"><text>CAS RN 59954-01-7</text></nplcit>), penbutolol sulfate (2-Propanol, l-(2-cyclopentylphenoxy)-3-[1,1-dimethyle- thyl)amino] 1 , (S)-, sulfate (2:1) (salt), <nplcit id="ncit0060" npl-type="c"><text>CAS RN 38363-32-5</text></nplcit>), practolol (Acetamide, N-[4-[2- hydroxy-3-[(1-methylethyl)amino]-propoxy]phenyl]-, <nplcit id="ncit0061" npl-type="c"><text>CAS RN 6673-35-4</text></nplcit>;) tiprenolol hydrochloride (Propanol, l-[(1-methylethyl)amino]-3-[2-(metylthio)-phenoxy]-, hydrochloride, (±), <nplcit id="ncit0062" npl-type="c"><text>CAS RN 39832-43-4</text></nplcit>), tolamolol (Benzamide, 4-[2-[[2-hydroxy-3-(2-methylphenoxy)-propyl] amino] ethoxyl]-, <nplcit id="ncit0063" npl-type="c"><text>CAS RN 38103-61-6</text></nplcit>), bopindolol, indenolol, pindolol, propanolol, tertatolol, and tilisolol, and the like; calcium channel blockers such as besylate salt of amlodipine (such as 3-ethyl-5-methyl-2-(2-aminoethoxymethyl)-4-(2-chlorophenyl)-l ,4-dihydro-6-methyl-3,5-pyridinedicarboxylate benzenesulphonate, e.g., Norvasc^®, Pfizer), clentiazem maleate (1,5-Benzothiazepin-4(5H)-one, 3-(acetyloxy)-8-chloro-5-[2-(dimethylamino)ethyl]-2,3-dihydro-2-(4-methoxyphenyl)-(2S-cis)-, (Z)-2-butenedioate (1 :1), see also <patcit id="pcit0649" dnum="US4567195A"><text>US4567195</text></patcit>), isradipine (3,5-Pyridinedicarboxylic acid, 4-(4-benzofurazanyl)-l,4-dihydro-2,6-dimethyl-, methyl 1-methylethyl ester, (±)-4(4-benzofurazanyl)- 1 ,4-dihydro-2,6-dimethyl-3 ,5 - pyridinedicarboxylate, see also <patcit id="pcit0650" dnum="US4466972A"><text>US4466972</text></patcit>); nimodipine (such as is isopropyl (2- methoxyethyl) 1, 4- dihydro -2,6- dimethyl -4- (3-nitrophenyl) -3,5- pyridine - dicarboxylate, e.g. Nimotop^®, Bayer), felodipine (such as ethyl methyl 4-(2,3-dichlorophetnyl)-l,4-dihydro-2,6-dimetthyl-3,5-pyridinedicarboxylate- , e.g. Plendil^® Extended-Release, AstraZeneca LP), nilvadipine (3,5-Pyridinedicarboxylic acid, 2-cyano-l,4-dihydro-6-methyl-4-(3-nitrophenyl)-,3-methyl 5-(1-methylethyl) ester, also see <patcit id="pcit0651" dnum="US3799934A"><text>US3799934</text></patcit>), nifedipine (such as 3, 5 -pyridinedicarboxylic acid,l,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-, dimethyl ester, e.g., Procardia XL^® Extended Release Tablets, Pfizer), diltiazem hydrochloride (such as l,5,-Benzothiazepin-4(5H)-one,3-(acetyloxy)-5[2-(dimethylamino)ethyl]-2,-3-dihydro-2(4-methoxyphenyl)-, monohydrochloride, (+)-cis., e.g., Tiazac^®, Forest), verapamil hydrochloride (such as benzeneacetronitrile, (alpha)-[[3-[[2-(3,4-dimethoxyphenyl) ethyl]methylamino]propyl] -3 ,4-dimethoxy-(alpha)-( 1 -methylethyl) hydrochloride, e.g., Isoptin^® SR, Knoll Labs), teiudipine hydrochloride (3,5-Pyridinedicarboxylic acid, 2-[(dimethylamino)methyl]4-[2-[(lE)-3-(1,1-dimethylethoxy)-3-oxo- l-propenyl]phenyl]-l,4-dihydro-6-methyl-, diethyl ester, monohydrochloride) <nplcit id="ncit0064" npl-type="c"><text>CAS RN 108700-03-4</text></nplcit>), belfosdil (Phosphonic acid, [2-(2-phenoxy ethyl)- 1,3 -propane- diyl]bis-, tetrabutyl ester <nplcit id="ncit0065" npl-type="c"><text>CAS RN 103486-79-9</text></nplcit>), fostedil (Phosphonic acid, [[4-(2-benzothiazolyl)phenyl]methyl]-, diethyl ester <nplcit id="ncit0066" npl-type="c"><text>CAS RN 75889-62-2</text></nplcit>), aranidipine, azelnidipine, bamidipine, benidipine, bepridil, cinaldipine, clevidipine, efonidipine, gallopamil, lacidipine, lemildipine, lercanidipine, monatepil maleate (1-Piperazinebutanamide, N-(6, 11 -dihydrodibenzo(b,e)thiepin- 11 -yl)4-(4-fluorophenyl)-, (+)-, (Z)-2-butenedioate (1 :1) (±)-N-(6,l l-Dihydrodibenzo(b,e)thiep- in-l l-yl)-4-(p- fluorophenyl)-1-piperazinebutyrarnide maleate (1 :1) <nplcit id="ncit0067" npl-type="c"><text>CAS RN 132046-06-1</text></nplcit>), nicardipine, nisoldipine, nitrendipine, manidipine, pranidipine, and the like; T-channel calcium antagonists such as mibefradil; angiotensin converting enzyme (ACE) inhibitors such as benazepril, benazepril hydrochloride (such as 3-[[l-(ethoxycarbonyl)-3- phenyl-( 1 S)-propyl]amino]-2,3 ,4,5-tetrahydro-2-oxo- 1 H - 1 -(3 S)-benzazepine- 1 -acetic acid monohydrochloride, e.g., Lotrel^®, Novartis), captopril (such as l-[(2S)-3-mercapto-2- methylpropioityl]-L-proline, e.g., Captopril, Mylan, <nplcit id="ncit0068" npl-type="c"><text>CAS RN 62571-86-2</text></nplcit> and others disclosed in <patcit id="pcit0652" dnum="US4046889A"><text>US4046889</text></patcit>), ceranapril (and others disclosed in <patcit id="pcit0653" dnum="US4452790A"><text>US4452790</text></patcit>), cetapril (alacepril, Dainippon disclosed in <nplcit id="ncit0069" npl-type="s"><text>Eur. Therap. Res. 39:671 (1986</text></nplcit>); 40:543 (1986)), cilazapril (Hoffman-LaRoche) disclosed in <nplcit id="ncit0070" npl-type="s"><text>J. Cardiovasc. Pharmacol. 9:39 (1987</text></nplcit>), indalapril (delapril hydrochloride (2H-1,2,4- Benzothiadiazine-7-sulfonamide, 3-bicyclo[2.2.1 ]hept-5-en-2-yl-6-chloro-3,4-dihydro-, 1,1- dioxide <nplcit id="ncit0071" npl-type="c"><text>CAS RN 2259-96-3</text></nplcit>); disclosed in <patcit id="pcit0654" dnum="US4385051A"><text>US4385051</text></patcit>), enalapril (and others disclosed in <patcit id="pcit0655" dnum="US4374829A"><text>US4374829</text></patcit>), enalopril, enaloprilat, fosinopril, ((such as L-proline, 4-cyclohexyl-1-[[[2-methyl- l-(1-oxopropoxy) propoxy](4-phenylbutyl) phosphinyl]acetyl]-, sodium salt, e.g., Monopril, Bristol-Myers Squibb and others disclosed in <patcit id="pcit0656" dnum="US4168267A"><text>US4168267</text></patcit>), fosinopril sodium (L- Proline, 4-cyclohexyl-1-[[(R)-[(lS)-2-rnetliyl-1-(1-ox- opropoxy)propox), imidapril, indolapril (Schering, disclosed in <nplcit id="ncit0072" npl-type="s"><text>J. Cardiovasc. Pharmacol. 5:643, 655 (1983</text></nplcit>)), lisinopril (Merck), losinopril, moexipril, moexipril hydrochloride (3-Isoquinolinecarboxylic acid, 2-[(2S)-2-[[(1S)- 1 -(ethoxycarbonyl)-3-phenylpropyl]amino]- 1 - oxopropyl]- 1 ,- 2,3,4-tetrahydro-6,7-dimethoxy-, monohydrochloride, (3S)- <nplcit id="ncit0073" npl-type="c"><text>CAS RN 82586-52-5</text></nplcit>), quinapril, quintprilat, ramipril (Hoechsst) disclosed in <patcit id="pcit0657" dnum="EP79022A"><text>EP 79022 </text></patcit>and <nplcit id="ncit0074" npl-type="s"><text>Curr. Ther. Res. 40:74 (1986</text></nplcit>), perindopril erbuinine (such as 2S,3aS,7aS- 1 -[(S)-N-[(S)- 1 - Carboxybutyljalanyljhexahydro^-indolinecarboxylic acid, 1 -ethyl ester, compound with tert-butylamine (1 :1), e.g., Aceon^®, Solvay), perindopril (Servier, disclosed in <nplcit id="ncit0075" npl-type="s"><text>Eur. J. clin. Pharmacol. 31 :519 (1987</text></nplcit>)), quanipril (disclosed in <patcit id="pcit0658" dnum="US4344949A"><text>US4344949</text></patcit>), spirapril (Schering, disclosed in <nplcit id="ncit0076" npl-type="s"><text>Acta. Pharmacol. Toxicol. 59 (Supp. 5): 173 (1986</text></nplcit>)), tenocapril, trandolapril, zofenopril (and others disclosed in <patcit id="pcit0659" dnum="US4316906A"><text>US4316906</text></patcit>), rentiapril (fentiapril, disclosed in <nplcit id="ncit0077" npl-type="s"><text>Clin. Exp. Pharmacol. Physiol. 10:131 (1983</text></nplcit>)), pivopril, YS980, teprotide (Bradykinin potentiator BPP9a <nplcit id="ncit0078" npl-type="c"><text>CAS RN 35115-60-7</text></nplcit>), BRL 36,378 (Smith Kline Beecham, see <patcit id="pcit0660" dnum="EP80822A"><text>EP80822</text></patcit> and <patcit id="pcit0661" dnum="EP60668A"><text>EP60668</text></patcit>), MC-838 (Chugai, see CA. 102:72588v and <nplcit id="ncit0079" npl-type="s"><text>Jap. J. Pharmacol. 40:373 (1986</text></nplcit>), CGS 14824 (Ciba-Geigy, 3-([l-ethoxycarbonyl-3-phenyl-(lS)-propyl]amino)-2,3,4,5-tetrahydro-2-ox- o-1-(3S)-benzazepine-l acetic acid HCl, see <patcit id="pcit0662" dnum="GB2103614A"><text>U.K. Patent No. 2103614</text></patcit>), CGS 16,617 (Ciba- Geigy, 3(S)-[[(lS)-5-amino-1-carboxypentyl]amino]-2,3,4,- 5-tetrahydro-2-oxo-1H-1- benzazepine-1-ethanoic acid, see <patcit id="pcit0663" dnum="US4473575A"><text>US4473575</text></patcit>), Ru 44570 (Hoechst, see <nplcit id="ncit0080" npl-type="s"><text>Arzneimittelforschung 34:1254 (1985</text></nplcit>)), R 31-2201 (<nplcit id="ncit0081" npl-type="s"><text>Hoffman-LaRoche see FEBS Lett. 165:201 (1984</text></nplcit>)), CI925 (<nplcit id="ncit0082" npl-type="s"><text>Pharmacologist 26:243, 266 (1984</text></nplcit>)), WY-44221 (<nplcit id="ncit0083" npl-type="s"><text>Wyeth, see J. Med. Chem. 26:394 (1983</text></nplcit>)), and those disclosed in <patcit id="pcit0664" dnum="US2003006922A"><text>US2003006922</text></patcit> (paragraph 28), <patcit id="pcit0665" dnum="US4337201A"><text>US4337201</text></patcit>, <patcit id="pcit0666" dnum="US4432971A"><text>US4432971</text></patcit> (phosphonamidates); neutral endopeptidase inhibitors such as omapatrilat (Vaniev^®), CGS 30440, cadoxatril and ecadotril, fasidotril (also known as aladotril or alatriopril), sampatrilat, mixanpril, and gemopatrilat, AVE7688, ER4030, and those disclosed in <patcit id="pcit0667" dnum="US5362727A"><text>US5362727</text></patcit>, <patcit id="pcit0668" dnum="US5366973A"><text>US5366973</text></patcit>, <patcit id="pcit0669" dnum="US5225401A"><text>US5225401</text></patcit>, <patcit id="pcit0670" dnum="US4722810A"><text>US4722810</text></patcit>, <patcit id="pcit0671" dnum="US5223516A"><text>US5223516</text></patcit>, <patcit id="pcit0672" dnum="US4749688A"><text>US4749688</text></patcit>, <patcit id="pcit0673" dnum="US5552397A"><text>US5552397</text></patcit>, <patcit id="pcit0674" dnum="US5504080A"><text>US5504080</text></patcit>, <patcit id="pcit0675" dnum="US5612359A"><text>US5612359</text></patcit>, <patcit id="pcit0676" dnum="US5525723A"><text>US5525723</text></patcit>, <patcit id="pcit0677" dnum="EP0599444A"><text>EP0599444</text></patcit>, <patcit id="pcit0678" dnum="EP0481522A"><text>EP0481522</text></patcit>, <patcit id="pcit0679" dnum="EP0599444A"><text>EP0599444</text></patcit>, <patcit id="pcit0680" dnum="EP0595610A"><text>EP0595610</text></patcit>, <patcit id="pcit0681" dnum="EP0534363A"><text>EP0534363</text></patcit>, <patcit id="pcit0682" dnum="EP534396A"><text>EP534396</text></patcit>, <patcit id="pcit0683" dnum="EP534492A"><text>EP534492</text></patcit>, <patcit id="pcit0684" dnum="EP0629627A"><text>EP0629627</text></patcit>; endothelin antagonists such as tezosentan, A308165, and YM62899, and the like; vasodilators such as hydralazine (apresoline), clonidine (clonidine hydrochloride (1H-Imidazol- 2-amine, N-(2,6-dichlorophenyl)4,5-dihydro-, monohydrochloride <nplcit id="ncit0084" npl-type="c"><text>CAS RN 4205-91-8</text></nplcit>), catapres, minoxidil (loniten), nicotinyl alcohol (roniacol), diltiazem hydrochloride (such as 1,5- Benzothiazepin-4(5H)-one,3-(acetyloxy)-5[2-(dimethylamino)ethyl]-2,-3-dihydro-2(4- methoxyphenyl)-, monohydrochloride, (+)-cis, e.g., Tiazac^®, Forest), isosorbide dinitrate (such as 1,4:3,6-dianhydro-D-glucitol 2,5-dinitrate e.g., Isordil^® Titradose^®, Wyeth- Ayerst), sosorbide mononitrate (such as 1,4:3,6-dianhydro-D-glucito- 1,5-nitrate, an organic nitrate, e.g., Ismo^® Wyeth-Ayerst), nitroglycerin (such as 2,3 propanetriol trinitrate, e.g., Nitrostat^® Parke- Davis), verapamil hydrochloride (such as benzeneacetonitrile, (±)-(alpha)[3-[[2-(3,4 dimethoxypheny l)ethyl]methylamino]propyl] -3 ,4-dimethoxy-(alpha)- ( 1 -methylethyl) hydrochloride, e.g., Covera HS^® Extended-Release, Searle), chromonar (which may be prepared as disclosed in <patcit id="pcit0685" dnum="US3282938A"><text>US3282938</text></patcit>), clonitate (Annalen 1870 155), droprenilamine (which may be prepared as disclosed in <patcit id="pcit0686" dnum="DE2521113"><text>DE2521113</text></patcit>), lidoflazine (which may be prepared as disclosed in <patcit id="pcit0687" dnum="US3267104A"><text>US3267104</text></patcit>); prenylamine (which may be prepared as disclosed in <patcit id="pcit0688" dnum="US3152173A"><text>US3152173</text></patcit>), propatyl nitrate (which may be prepared as disclosed in <patcit id="pcit0689" dnum="FR1103113"><text>French Patent No. 1,103,113</text></patcit>), mioflazine hydrochloride (1 -Piperazineacetamide, 3-(aminocarbonyl)4-[4,4-bis(4-fluorophenyl)butyl]-N-(2,6- dichlorophenyl)-, dihydrochloride <nplcit id="ncit0085" npl-type="c"><text>CAS RN 83898-67-3</text></nplcit>), mixidine (Benzeneethanamine, 3,4- dimethoxy-N-(1-methyl-2-pyrrolidinylidene)- Pyrrolidine, 2-[(3,4-dimethoxyphenethyl)imino]- 1 -methyl- l-Methyl-2- [(3, 4-dimethoxyphenethyl)imino]pyrrolidine <nplcit id="ncit0086" npl-type="c"><text>CAS RN 27737-38-8</text></nplcit>), molsidomine (1,2,3-Oxadiazolium, 5-[(ethoxycarbonyl)amino]-3-(4-morpholinyl)-, inner salt <nplcit id="ncit0087" npl-type="c"><text>CAS RN 25717-80-0</text></nplcit>), isosorbide mononitrate (D-Glucitol, l,4:3,6-dianhydro-, 5-nitrate <nplcit id="ncit0088" npl-type="c"><text>CAS RN 16051-77-7</text></nplcit>), erythrityl tetranitrate (1,2,3,4-Butanetetrol, tetranitrate, (2R,3S)-rel-<nplcit id="ncit0089" npl-type="c"><text>CAS RN 7297-25-8</text></nplcit>), clonitrate(l,2-Propanediol, 3-chloro-, dinitrate (7CI, 8CI, 9CI) <nplcit id="ncit0090" npl-type="c"><text>CAS RN 2612-33-1</text></nplcit>), dipyridamole Ethanol, 2,2',2",2'"-[(4,8-di-1-piperidinylpyrimido[5,4-d]pyrimidine-2,6-diyl)dinitrilo]tetrakis- <nplcit id="ncit0091" npl-type="c"><text>CAS RN 58-32-2</text></nplcit>), nicorandil (<nplcit id="ncit0092" npl-type="c"><text>CAS RN 65141-46-0 3</text></nplcit>-), pyridinecarboxamide (N-[2-(nitrooxy)ethyl]-Nisoldipine3,5-Pyridinedicarboxylic acid, 1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-, methyl 2-methylpropyl ester <nplcit id="ncit0093" npl-type="c"><text>CAS RN 63675-72-9</text></nplcit>), nifedipine3,5-Pyridinedicarboxylic acid, 1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-, dimethyl ester <nplcit id="ncit0094" npl-type="c"><text>CAS RN 21829-25-4</text></nplcit>), perhexiline maleate (Piperidine, 2-(2,2-dicyclohexylethyl)-, (2Z)-2-butenedioate (1 :1) <nplcit id="ncit0095" npl-type="c"><text>CAS RN 6724-53-4</text></nplcit>), oxprenolol hydrochloride (2-Propanol, 1-[(1-methylethyl)amino]-3-[2-(2-propenyloxy)phenoxy]-, hydrochloride <nplcit id="ncit0096" npl-type="c"><text>CAS RN 6452-73-9</text></nplcit>), pentrinitrol (1,3-Propanediol, 2,2-bis[(nitrooxy)methyl]-, mononitrate (ester) <nplcit id="ncit0097" npl-type="c"><text>CAS RN 1607-17-6</text></nplcit>), verapamil (Benzeneacetonitrile, α-[3-[[2-(3,4-dimethoxyphenyl)ethyl]- methylamino]propyl]-3, 4-dimethoxy-α-(l -methylethyl)- <nplcit id="ncit0098" npl-type="c"><text>CAS RN 52-53-9</text></nplcit>) and the like; angiotensin II receptor antagonists such as, aprosartan, zolasartan, olmesartan, pratosartan, FI6828K, RNH6270, candesartan (1 H-Benzimidazole-7-carboxylic acid, 2-ethoxy-1-[[2'-tetrazol-5-yl)[1,1-biphenyl]4-yl]methyl]- <nplcit id="ncit0099" npl-type="c"><text>CAS RN 139481-59-7</text></nplcit>), candesartan cilexetil ((+/-)-1-(cyclohexylcarbonyloxy)ethyl-2-ethoxy-1-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]-1H-benzimidazole carboxylate, <nplcit id="ncit0100" npl-type="c"><text>CAS RN 145040-37-5</text></nplcit>, <patcit id="pcit0690" dnum="US5703110A"><text>US5703110</text></patcit> and <patcit id="pcit0691" dnum="US5196444A"><text>US5196444</text></patcit>), eprosartan (3-[l-4-carboxyphenylmethyl)-2-n-butyl-imidazol-5-yl]-(2-thienylmethyl) propenoic acid, <patcit id="pcit0692" dnum="US5185351A"><text>US5185351</text></patcit> and <patcit id="pcit0693" dnum="US5650650A"><text>US5650650</text></patcit>), irbesartan (2-n-butyl-3- [[2'-(lh-tetrazol-5-yl)biphenyl-4-yl]methyl] 1 ,3-diazazspiro[4,4]non-1-en-4-one, <patcit id="pcit0694" dnum="US5270317A"><text>US5270317</text></patcit> and <patcit id="pcit0695" dnum="US5352788A"><text>US5352788</text></patcit>), losartan (2-N-butyl-4-chloro-5-hydroxymethyl-1-[(2'-(1H-tetrazol-5-yl)biphenyl-4-yl)-methyl]imidazole, potassium salt, <patcit id="pcit0696" dnum="US5138069A"><text>US5138069</text></patcit>, <patcit id="pcit0697" dnum="US5153197A"><text>US5153197</text></patcit> and <patcit id="pcit0698" dnum="US5128355A"><text>US5128355</text></patcit>), tasosartan (5,8-dihydro-2,4-dimethyl-8-[(2'-(1H-tetrazol-5-yl)[1,r-biphenyl]4-yl)methyl]-pyrido[2,3-d]pyrimidin-7(6H)-one, <patcit id="pcit0699" dnum="US5149699A"><text>US5149699</text></patcit>), telmisartan (4'-[(1 ,4-dimethyl-2'-propyl-(2,6'-bi-1H-benzimidazol)-r-yl)]-[ 1 , 1 '-biphenyl]-2-carboxylic acid, <nplcit id="ncit0101" npl-type="c"><text>CAS RN 144701-48-4</text></nplcit>, <patcit id="pcit0700" dnum="US5591762A"><text>US5591762</text></patcit>), milfasartan, abitesartan, valsartan (Diovan^® (Novartis), (S)-N-valeryl-N-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]valine, <patcit id="pcit0701" dnum="US5399578A"><text>US5399578</text></patcit>), EXP-3137 (2-N-butyl-4-chloro-1-[(2'-(1H-tetrazol-5-yl)biphenyl-4-yl)-methyl]imidazole-5-carboxylic acid, <patcit id="pcit0702" dnum="US5138069A"><text>US5138069</text></patcit>, <patcit id="pcit0703" dnum="US513197A"><text>US513197</text></patcit> and <patcit id="pcit0704" dnum="US5128355A"><text>US5128355</text></patcit>), 3-(2'-(tetrazol-5-yl)-l,r-biphen-4-yl)methyl-5,7-dimethyl-2-ethyl-3H-imidazo[4,5-b]pyridine, 4'[2-ethyl-4-methyl-6-(5,6,7,8-tetrahydroimidazo[l,2-a]pyridin-2-yl]-benzimidazol-1-yl]-methyl]-l,r-biphenyl]-2- carboxylic acid, 2-butyl-6-(1-methoxy-1-methylethyl)-2-[2'-)IH-tetrazol-5-yl)biphenyl-4-ylmethyl] guinazolin-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]imidazole-carboxylic acid, 2-butyl-4-chloro-1-[[2'-(1H-1-tetrazol-5- yl) [ 1 , 1 ' -biphenyl] -4-yl]methyl]- 1 H-imidazole-5 -carboxylic acid- 1 -(ethoxycarbonyl-oxy)ethyl ester potassium salt, dipotassium 2-butyl-4-(methylthio)-1-[[2-[[[(propylamino)carbonyl]amino]-sulfonyl](1,1 '-biphenyl)-4-yl]methyl]-l H-imidazole-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-thiophencarboxylate, 5-[(3,5-dibutyl-1H-l,2,4-triazol-1-yl)methyl]-2-[2- ( 1 H-tetrazol-5 - ylphenyl)]pyridine, 6-butyl-2-(2-phenylethyl)-5 [[2'-(I H-tetrazol-5 -yl)[ 1,1 '- biphenyl]-4-methyl]pyrimidin-4-(3H)-one D,L lysine salt, 5-methyl-7-n-propyl-8-[[2'-(1H- tetrazol-5-yl)biphenyl-4-yl]methyl]-[ 1 ,2,4]-triazolo[ 1 ,5-c]pyrimidin-2(3H)-one, 2,7-diethyl-5- [[2'-(5-tetrazoly)biphenyl-4-yl]methyl]-5H-pyrazolo[l,5-b][l,2,4]triazole potassium salt, 2-[2- butyl-4,5-dihydro-4-oxo-3-[2'-(1H-tetrazol-5-yl)-4-biphenylmethyl]-3H-imidazol[4,5- c]pyridine-5-ylmethyl]benzoic acid, ethyl ester, potassium salt, 3-methoxy-2,6-dimethyl-4- [[2'(1H-tetrazol-5-yl)-1,1'-bipheityl-4-yl]methoxy]pyridine, 2-ethoxy-1-[[2'-(5-oxo-2,5-dihydro- 1 ,2,4-oxadiazol-3 - yl)biphenyl-4-yl]methyl] - 1 H-beiizimidazole-7-carboxylic acid, 1 - [N-(2'-( 1H- tetrazol-5-yl)biphenyl-4-yl-methyl)-N-valerolylaminomethyl)cyclopentane- 1 -carboxylic acid, 7- methyl-2n-propyl-3-[[2' 1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-3H-imidazo[4,5-6]pyridine, 2- [5-[(2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridine-3-yl)methyl]-2-quinolinyl]sodium benzoate, 2-butyl-6-chloro-4-hydroxymethyl-5 -methyl-3 -[[2'-(I H-tetrazol-5 -yl)biphenyl-4-yl]methyl]pyridine, 2- [ [[2-butyl- 1 - [(4-carboxyphenyl)methyl] - 1 H-imidazol-5 - yl]methyl]amino]benzoic acid tetrazol-5-yl)biphenyl-4-yl]methyl]pyrimidin-6-one, 4(S)- [4-(carboxymethyl)phenoxy]-N-[2(R)-[4-(2-sulfobenzamido)imidazol- 1 -yl]octanoyl]-L-proline, 1 - (2,6-dimethylphenyl)-4-butyl-l,3-dihydro-3-[[6-[2-(1H-tetrazol-5-yl)phenyl]-3-pyridinyl]methyl]-2H-imidazol-2-one, 5 ,8-ethano-5 ,8-dimethyl-2-n-propyl-5 ,6,7,8-tetrahydro-1 - [[2'(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-1H,4H-l,3,4a,8a-tetrazacyclopentanaphthalene-9-one, 4-[1-[2'-(l,2,3,4-tetrazol-5-yl)biphen-4-yl)methylammo]-5,6,7,8-tetrahydro-2-trifylquinazoline, 2-(2-chlorobenzoyl)imino-5-ethyl-3-[2'-(1H-tetrazole-5-yl)biphenyl-4-yl)niethyl-l,3,4-thiadiazoline, 2-[5-ethyl-3-[2-(1H-tetrazole-5-yl)biphenyl-4-yl]methyl-l,3,4-thiazoline-2-ylidene]aminocarbonyl-1-cyclopentencarboxylic acid dipotassium salt, and 2-butyl-4-[N-methyl-N-(3 -methylcrotonoyl)amino] - 1 - [ [2'-( 1 H-tetrazol-5 -yl)biphenyl-4-yl]methyl]- 1 H- imidzole-5 -carboxylic acid 1-ethoxycarbonyloxyethyl ester, those disclosed in patent publications <patcit id="pcit0705" dnum="EP475206A"><text>EP475206</text></patcit>, <patcit id="pcit0706" dnum="EP497150A"><text>EP497150</text></patcit>, <patcit id="pcit0707" dnum="EP539086A"><text>EP539086</text></patcit>, <patcit id="pcit0708" dnum="EP539713A"><text>EP539713</text></patcit>, <patcit id="pcit0709" dnum="EP535463A"><text>EP535463</text></patcit>, <patcit id="pcit0710" dnum="EP535465A"><text>EP535465</text></patcit>, <patcit id="pcit0711" dnum="EP542059A"><text>EP542059</text></patcit>, <patcit id="pcit0712" dnum="EP497121A"><text>EP497121</text></patcit>, <patcit id="pcit0713" dnum="EP535420A"><text>EP535420</text></patcit>, <patcit id="pcit0714" dnum="EP407342A"><text>EP407342</text></patcit>, <patcit id="pcit0715" dnum="EP415886A"><text>EP415886</text></patcit>, <patcit id="pcit0716" dnum="EP424317A"><text>EP424317</text></patcit>, <patcit id="pcit0717" dnum="EP435827A"><text>EP435827</text></patcit>, <patcit id="pcit0718" dnum="EP433983A"><text>EP433983</text></patcit>, <patcit id="pcit0719" dnum="EP475898A"><text>EP475898</text></patcit>, <patcit id="pcit0720" dnum="EP490820A"><text>EP490820</text></patcit>, <patcit id="pcit0721" dnum="EP528762A"><text>EP528762</text></patcit>, <patcit id="pcit0722" dnum="EP324377A"><text>EP324377</text></patcit>, <patcit id="pcit0723" dnum="EP323841A"><text>EP323841</text></patcit>, <patcit id="pcit0724" dnum="EP420237A"><text>EP420237</text></patcit>, <patcit id="pcit0725" dnum="EP500297A"><text>EP500297</text></patcit>, <patcit id="pcit0726" dnum="EP426021A"><text>EP426021</text></patcit>, <patcit id="pcit0727" dnum="EP480204A"><text>EP480204</text></patcit>, <patcit id="pcit0728" dnum="EP429257A"><text>EP429257</text></patcit>, <patcit id="pcit0729" dnum="EP430709A"><text>EP430709</text></patcit>, <patcit id="pcit0730" dnum="EP434249A"><text>EP434249</text></patcit>, <patcit id="pcit0731" dnum="EP446062A"><text>EP446062</text></patcit>, <patcit id="pcit0732" dnum="EP505954A"><text>EP505954</text></patcit>, <patcit id="pcit0733" dnum="EP524217A"><text>EP524217</text></patcit>, <patcit id="pcit0734" dnum="EP514197A"><text>EP514197</text></patcit>, <patcit id="pcit0735" dnum="EP514198A"><text>EP514198</text></patcit>, <patcit id="pcit0736" dnum="EP514193A"><text>EP514193</text></patcit>, <patcit id="pcit0737" dnum="EP514192A"><text>EP514192</text></patcit>, <patcit id="pcit0738" dnum="EP450566A"><text>EP450566</text></patcit>, <patcit id="pcit0739" dnum="EP468372A"><text>EP468372</text></patcit>, <patcit id="pcit0740" dnum="EP485929A"><text>EP485929</text></patcit>, <patcit id="pcit0741" dnum="EP503162A"><text>EP503162</text></patcit>, <patcit id="pcit0742" dnum="EP533058A"><text>EP533058</text></patcit>, <patcit id="pcit0743" dnum="EP467207A"><text>EP467207</text></patcit><patcit id="pcit0744" dnum="EP399731A"><text>EP399731</text></patcit>, <patcit id="pcit0745" dnum="EP399732A"><text>EP399732</text></patcit>, <patcit id="pcit0746" dnum="EP412848A"><text>EP412848</text></patcit>, <patcit id="pcit0747" dnum="EP453210A"><text>EP453210</text></patcit>, <patcit id="pcit0748" dnum="EP456442A"><text>EP456442</text></patcit>, <patcit id="pcit0749" dnum="EP470794A"><text>EP470794</text></patcit>, <patcit id="pcit0750" dnum="EP470795A"><text>EP470795</text></patcit>, <patcit id="pcit0751" dnum="EP495626A"><text>EP495626</text></patcit>, <patcit id="pcit0752" dnum="EP495627A"><text>EP495627</text></patcit>, <patcit id="pcit0753" dnum="EP499414A"><text>EP499414</text></patcit>, <patcit id="pcit0754" dnum="EP499416A"><text>EP499416</text></patcit>, <patcit id="pcit0755" dnum="EP499415A"><text>EP499415</text></patcit>, <patcit id="pcit0756" dnum="EP511791A"><text>EP511791</text></patcit>, <patcit id="pcit0757" dnum="EP516392A"><text>EP516392</text></patcit>, <patcit id="pcit0758" dnum="EP520723A"><text>EP520723</text></patcit>, <patcit id="pcit0759" dnum="EP520724A"><text>EP520724</text></patcit>, <patcit id="pcit0760" dnum="EP539066A"><text>EP539066</text></patcit>, <patcit id="pcit0761" dnum="EP438869A"><text>EP438869</text></patcit>, <patcit id="pcit0762" dnum="EP505893A"><text>EP505893</text></patcit>, <patcit id="pcit0763" dnum="EP530702A"><text>EP530702</text></patcit>, <patcit id="pcit0764" dnum="EP400835A"><text>EP400835</text></patcit>, <patcit id="pcit0765" dnum="EP400974A"><text>EP400974</text></patcit>, <patcit id="pcit0766" dnum="EP401030A"><text>EP401030</text></patcit>, <patcit id="pcit0767" dnum="EP407102A"><text>EP407102</text></patcit>, <patcit id="pcit0768" dnum="EP411766A"><text>EP411766</text></patcit>, <patcit id="pcit0769" dnum="EP409332A"><text>EP409332</text></patcit>, <patcit id="pcit0770" dnum="EP412594A"><text>EP412594</text></patcit>, <patcit id="pcit0771" dnum="EP419048A"><text>EP419048</text></patcit>, <patcit id="pcit0772" dnum="EP480659A"><text>EP480659</text></patcit>, <patcit id="pcit0773" dnum="EP481614A"><text>EP481614</text></patcit>, <patcit id="pcit0774" dnum="EP490587A"><text>EP490587</text></patcit>, <patcit id="pcit0775" dnum="EP467715A"><text>EP467715</text></patcit>, <patcit id="pcit0776" dnum="EP479479A"><text>EP479479</text></patcit>, <patcit id="pcit0777" dnum="EP502725A"><text>EP502725</text></patcit>, <patcit id="pcit0778" dnum="EP503838A"><text>EP503838</text></patcit>, <patcit id="pcit0779" dnum="EP505098A"><text>EP505098</text></patcit>, <patcit id="pcit0780" dnum="EP505111A"><text>EP505111</text></patcit><patcit id="pcit0781" dnum="EP513979A"><text>EP513,979</text></patcit><patcit id="pcit0782" dnum="EP507594A"><text>EP507594</text></patcit>, <patcit id="pcit0783" dnum="EP510812A"><text>EP510812</text></patcit>, <patcit id="pcit0784" dnum="EP511767A"><text>EP511767</text></patcit>, <patcit id="pcit0785" dnum="EP512675A"><text>EP512675</text></patcit>, <patcit id="pcit0786" dnum="EP512676A"><text>EP512676</text></patcit>, <patcit id="pcit0787" dnum="EP512870A"><text>EP512870</text></patcit>, <patcit id="pcit0788" dnum="EP517357A"><text>EP517357</text></patcit>, <patcit id="pcit0789" dnum="EP537937A"><text>EP537937</text></patcit>, <patcit id="pcit0790" dnum="EP534706A"><text>EP534706</text></patcit>, <patcit id="pcit0791" dnum="EP527534A"><text>EP527534</text></patcit>, <patcit id="pcit0792" dnum="EP540356A"><text>EP540356</text></patcit>, <patcit id="pcit0793" dnum="EP461040A"><text>EP461040</text></patcit>, <patcit id="pcit0794" dnum="EP540039A"><text>EP540039</text></patcit>, <patcit id="pcit0795" dnum="EP465368A"><text>EP465368</text></patcit>, <patcit id="pcit0796" dnum="EP498723A"><text>EP498723</text></patcit>, <patcit id="pcit0797" dnum="EP498722A"><text>EP498722</text></patcit>, <patcit id="pcit0798" dnum="EP498721A"><text>EP498721</text></patcit>, <patcit id="pcit0799" dnum="EP515265A"><text>EP515265</text></patcit>, <patcit id="pcit0800" dnum="EP503785A"><text>EP503785</text></patcit>, <patcit id="pcit0801" dnum="EP501892A"><text>EP501892</text></patcit>, <patcit id="pcit0802" dnum="EP519831A"><text>EP519831</text></patcit>, <patcit id="pcit0803" dnum="EP532410A"><text>EP532410</text></patcit>, <patcit id="pcit0804" dnum="EP498361A"><text>EP498361</text></patcit>, <patcit id="pcit0805" dnum="EP432737A"><text>EP432737</text></patcit>, <patcit id="pcit0806" dnum="EP504888A"><text>EP504888</text></patcit>, <patcit id="pcit0807" dnum="EP508393A"><text>EP508393</text></patcit>, <patcit id="pcit0808" dnum="EP508445A"><text>EP508445</text></patcit>, <patcit id="pcit0809" dnum="EP403159A"><text>EP403159</text></patcit>, <patcit id="pcit0810" dnum="EP403158A"><text>EP403158</text></patcit>, <patcit id="pcit0811" dnum="EP425211A"><text>EP425211</text></patcit>, <patcit id="pcit0812" dnum="EP427463A"><text>EP427463</text></patcit>, <patcit id="pcit0813" dnum="EP437103A"><text>EP437103</text></patcit>, <patcit id="pcit0814" dnum="EP481448A"><text>EP481448</text></patcit>, <patcit id="pcit0815" dnum="EP488532A"><text>EP488532</text></patcit>, <patcit id="pcit0816" dnum="EP501269A"><text>EP501269</text></patcit>, <patcit id="pcit0817" dnum="EP500409A"><text>EP500409</text></patcit>, <patcit id="pcit0818" dnum="EP540400A"><text>EP540400</text></patcit>, <patcit id="pcit0819" dnum="EP005528A"><text>EP005528</text></patcit>, <patcit id="pcit0820" dnum="EP028834A"><text>EP028834</text></patcit>, <patcit id="pcit0821" dnum="EP028833A"><text>EP028833</text></patcit>, <patcit id="pcit0822" dnum="EP411507A"><text>EP411507</text></patcit>, <patcit id="pcit0823" dnum="EP425921A"><text>EP425921</text></patcit>, <patcit id="pcit0824" dnum="EP430300A"><text>EP430300</text></patcit>, <patcit id="pcit0825" dnum="EP434038A"><text>EP434038</text></patcit>, <patcit id="pcit0826" dnum="EP442473A"><text>EP442473</text></patcit>, <patcit id="pcit0827" dnum="EP443568A"><text>EP443568</text></patcit>, <patcit id="pcit0828" dnum="EP445811A"><text>EP445811</text></patcit>, <patcit id="pcit0829" dnum="EP459136A"><text>EP459136</text></patcit>, <patcit id="pcit0830" dnum="EP483683A"><text>EP483683</text></patcit>, <patcit id="pcit0831" dnum="EP518033A"><text>EP518033</text></patcit>, <patcit id="pcit0832" dnum="EP520423A"><text>EP520423</text></patcit>, <patcit id="pcit0833" dnum="EP531876A"><text>EP531876</text></patcit>, <patcit id="pcit0834" dnum="EP531874A"><text>EP531874</text></patcit>, <patcit id="pcit0835" dnum="EP392317A"><text>EP392317</text></patcit>, <patcit id="pcit0836" dnum="EP468470A"><text>EP468470</text></patcit>, <patcit id="pcit0837" dnum="EP470543A"><text>EP470543</text></patcit>, <patcit id="pcit0838" dnum="EP502314A"><text>EP502314</text></patcit>, <patcit id="pcit0839" dnum="EP529253A"><text>EP529253</text></patcit>, <patcit id="pcit0840" dnum="EP543263A"><text>EP543263</text></patcit>, <patcit id="pcit0841" dnum="EP540209A"><text>EP540209</text></patcit>, <patcit id="pcit0842" dnum="EP449699A"><text>EP449699</text></patcit>, <patcit id="pcit0843" dnum="EP465323A"><text>EP465323</text></patcit>, <patcit id="pcit0844" dnum="EP521768A"><text>EP521768</text></patcit>, <patcit id="pcit0845" dnum="EP415594A"><text>EP415594</text></patcit>, <patcit id="pcit0846" dnum="WO9214468A"><text>WO92/14468</text></patcit>, <patcit id="pcit0847" dnum="WO9308171A"><text>WO93/08171</text></patcit>, <patcit id="pcit0848" dnum="WO9308169A"><text>WO93/08169</text></patcit>, <patcit id="pcit0849" dnum="WO9100277A"><text>WO91/00277</text></patcit>, <patcit id="pcit0850" dnum="WO9100281A"><text>WO91/00281</text></patcit>, <patcit id="pcit0851" dnum="WO9114367A"><text>WO91/14367</text></patcit>, <patcit id="pcit0852" dnum="WO9200067A"><text>WO92/00067</text></patcit>, <patcit id="pcit0853" dnum="WO9200977A"><text>WO92/00977</text></patcit>, <patcit id="pcit0854" dnum="WO9220342A"><text>WO92/20342</text></patcit>, <patcit id="pcit0855" dnum="WO9304045A"><text>WO93/04045</text></patcit>, <patcit id="pcit0856" dnum="WO9304046A"><text>WO93/04046</text></patcit>, <patcit id="pcit0857" dnum="WO9115206A"><text>WO91/15206</text></patcit>, <patcit id="pcit0858" dnum="WO9214714A"><text>WO92/14714</text></patcit>, <patcit id="pcit0859" dnum="WO9209600A"><text>WO92/09600</text></patcit>, <patcit id="pcit0860" dnum="WO9216552A"><text>WO92/16552</text></patcit>, <patcit id="pcit0861" dnum="WO9305025A"><text>WO93/05025</text></patcit>, <patcit id="pcit0862" dnum="WO9303018A"><text>WO93/03018</text></patcit>, <patcit id="pcit0863" dnum="WO9107404A"><text>WO91/07404</text></patcit>, <patcit id="pcit0864" dnum="WO9202508A"><text>WO92/02508</text></patcit>, <patcit id="pcit0865" dnum="WO9213853A"><text>WO92/13853</text></patcit>, <patcit id="pcit0866" dnum="WO9119697A"><text>WO91/19697</text></patcit>, <patcit id="pcit0867" dnum="WO9111909A"><text>WO91/11909</text></patcit>, <patcit id="pcit0868" dnum="WO9112001A"><text>WO91/12001</text></patcit>, <patcit id="pcit0869" dnum="WO9111999A"><text>WO91/11999</text></patcit>, <patcit id="pcit0870" dnum="WO9115209A"><text>WO91/15209</text></patcit>, <patcit id="pcit0871" dnum="WO9115479A"><text>WO91/15479</text></patcit>, <patcit id="pcit0872" dnum="WO9220687A"><text>WO92/20687</text></patcit>, <patcit id="pcit0873" dnum="WO9220662A"><text>WO92/20662</text></patcit>, <patcit id="pcit0874" dnum="WO9220661A"><text>WO92/20661</text></patcit>, <patcit id="pcit0875" dnum="WO9301177A"><text>WO93/01177</text></patcit>, <patcit id="pcit0876" dnum="WO9114679A"><text>WO91/14679</text></patcit>, <patcit id="pcit0877" dnum="WO9113063A"><text>WO91/13063</text></patcit>, <patcit id="pcit0878" dnum="WO9213564A"><text>WO92/13564</text></patcit>, <patcit id="pcit0879" dnum="WO9117148A"><text>WO91/17148</text></patcit>, <patcit id="pcit0880" dnum="WO9118888A"><text>WO91/18888</text></patcit>, <patcit id="pcit0881" dnum="WO9119715A"><text>WO91/19715</text></patcit>, <patcit id="pcit0882" dnum="WO9202257A"><text>WO92/02257</text></patcit>, <patcit id="pcit0883" dnum="WO9204335A"><text>WO92/04335</text></patcit>, <patcit id="pcit0884" dnum="WO9205161A"><text>WO92/05161</text></patcit>, <patcit id="pcit0885" dnum="WO9207852A"><text>WO92/07852</text></patcit>, <patcit id="pcit0886" dnum="WO9215577A"><text>WO92/15577</text></patcit>, <patcit id="pcit0887" dnum="WO9303033A"><text>WO93/03033</text></patcit>), <patcit id="pcit0888" dnum="WO9116313A"><text>WO91/16313</text></patcit>, <patcit id="pcit0889" dnum="WO9200068A"><text>WO92/00068</text></patcit>, <patcit id="pcit0890" dnum="WO9202510A"><text>WO92/02510</text></patcit>, <patcit id="pcit0891" dnum="WO9209278A"><text>WO92/09278</text></patcit>, <patcit id="pcit0892" dnum="WO9210179A"><text>WO9210179</text></patcit>, <patcit id="pcit0893" dnum="WO9210180A"><text>WO92/10180</text></patcit>, <patcit id="pcit0894" dnum="WO9210186A"><text>WO92/10186</text></patcit>, <patcit id="pcit0895" dnum="WO9210181A"><text>WO92/10181</text></patcit>, <patcit id="pcit0896" dnum="WO9210097A"><text>WO92/10097</text></patcit>, <patcit id="pcit0897" dnum="WO9210183A"><text>WO92/10183</text></patcit>, <patcit id="pcit0898" dnum="WO9210182A"><text>WO92/10182</text></patcit>, <patcit id="pcit0899" dnum="WO9210187A"><text>WO92/10187</text></patcit>, <patcit id="pcit0900" dnum="WO9210184A"><text>WO92/10184</text></patcit>, <patcit id="pcit0901" dnum="WO9210188A"><text>WO92/10188</text></patcit>, <patcit id="pcit0902" dnum="WO9210180A"><text>WO92/10180</text></patcit>, <patcit id="pcit0903" dnum="WO9210185A"><text>WO92/10185</text></patcit>, <patcit id="pcit0904" dnum="WO9220651A"><text>WO92/20651</text></patcit>, <patcit id="pcit0905" dnum="WO9303722A"><text>WO93/03722</text></patcit>, <patcit id="pcit0906" dnum="WO9306828A"><text>WO93/06828</text></patcit>, <patcit id="pcit0907" dnum="WO9303040A"><text>WO93/03040</text></patcit>, <patcit id="pcit0908" dnum="WO9219211A"><text>WO92/19211</text></patcit>, <patcit id="pcit0909" dnum="WO9222533A"><text>WO92/22533</text></patcit>, <patcit id="pcit0910" dnum="WO9206081A"><text>WO92/06081</text></patcit>, <patcit id="pcit0911" dnum="WO9205784A"><text>WO92/05784</text></patcit>, <patcit id="pcit0912" dnum="WO9300341A"><text>WO93/00341</text></patcit>, <patcit id="pcit0913" dnum="WO9204343A"><text>WO92/04343</text></patcit>, <patcit id="pcit0914" dnum="WO9204059A"><text>WO92/04059</text></patcit>, <patcit id="pcit0915" dnum="US5104877A"><text>US5104877</text></patcit>, <patcit id="pcit0916" dnum="US5187168A"><text>US5187168</text></patcit>, <patcit id="pcit0917" dnum="US5149699A"><text>US5149699</text></patcit>, <patcit id="pcit0918" dnum="US5185340A"><text>US5185340</text></patcit>, <patcit id="pcit0919" dnum="US4880804A"><text>US4880804</text></patcit>, <patcit id="pcit0920" dnum="US5138069A"><text>US5138069</text></patcit>, <patcit id="pcit0921" dnum="US4916129A"><text>US4916129</text></patcit>, <patcit id="pcit0922" dnum="US5153197A"><text>US5153197</text></patcit>, <patcit id="pcit0923" dnum="US5173494A"><text>US5173494</text></patcit>, <patcit id="pcit0924" dnum="US5137906A"><text>US5137906</text></patcit>, <patcit id="pcit0925" dnum="US5155126A"><text>US5155126</text></patcit>, <patcit id="pcit0926" dnum="US5140037A"><text>US5140037</text></patcit>, <patcit id="pcit0927" dnum="US5137902A"><text>US5137902</text></patcit>, <patcit id="pcit0928" dnum="US5157026A"><text>US5157026</text></patcit>, <patcit id="pcit0929" dnum="US5053329A"><text>US5053329</text></patcit>, <patcit id="pcit0930" dnum="US5132216A"><text>US5132216</text></patcit>, <patcit id="pcit0931" dnum="US5057522A"><text>US5057522</text></patcit>, <patcit id="pcit0932" dnum="US5066586A"><text>US5066586</text></patcit>, <patcit id="pcit0933" dnum="US5089626A"><text>US5089626</text></patcit>, <patcit id="pcit0934" dnum="US5049565A"><text>US5049565</text></patcit>, <patcit id="pcit0935" dnum="US5087702A"><text>US5087702</text></patcit>, <patcit id="pcit0936" dnum="US5124335A"><text>US5124335</text></patcit>, <patcit id="pcit0937" dnum="US5102880A"><text>US5102880</text></patcit>, <patcit id="pcit0938" dnum="US51128327B"><text>US51128327</text></patcit>, <patcit id="pcit0939" dnum="US5151435A"><text>US5151435</text></patcit>, <patcit id="pcit0940" dnum="US5202322A"><text>US5202322</text></patcit>, <patcit id="pcit0941" dnum="US5187159A"><text>US5187159</text></patcit>, <patcit id="pcit0942" dnum="US5198438A"><text>US5198438</text></patcit>, <patcit id="pcit0943" dnum="US5182288A"><text>US5182288</text></patcit>, <patcit id="pcit0944" dnum="US5036048A"><text>US5036048</text></patcit>, <patcit id="pcit0945" dnum="US5140036A"><text>US5140036</text></patcit>, <patcit id="pcit0946" dnum="US5087634A"><text>US5087634</text></patcit>, <patcit id="pcit0947" dnum="US5196537A"><text>US5196537</text></patcit>, <patcit id="pcit0948" dnum="US5153347A"><text>US5153347</text></patcit>, <patcit id="pcit0949" dnum="US5191086A"><text>US5191086</text></patcit>, <patcit id="pcit0950" dnum="US5190942A"><text>US5190942</text></patcit>, <patcit id="pcit0951" dnum="US5177097A"><text>US5177097</text></patcit>, <patcit id="pcit0952" dnum="US5212177A"><text>US5212177</text></patcit>, <patcit id="pcit0953" dnum="US5208234A"><text>US5208234</text></patcit>, <patcit id="pcit0954" dnum="US5208235A"><text>US5208235</text></patcit>, <patcit id="pcit0955" dnum="US5212195A"><text>US5212195</text></patcit>, <patcit id="pcit0956" dnum="US5130439A"><text>US5130439</text></patcit>, <patcit id="pcit0957" dnum="US5045540A"><text>US5045540</text></patcit>, <patcit id="pcit0958" dnum="US5041152A"><text>US5041152</text></patcit>, and <patcit id="pcit0959" dnum="US5210204A"><text>US5210204</text></patcit>, and pharmaceutically acceptable salts and esters thereof, α/β adrenergic blockers such as nipradilol, arotinolol, amosulalol, bretylium tosylate (<nplcit id="ncit0102" npl-type="c"><text>CAS RN: 61-75-6</text></nplcit>), dihydroergtamine mesylate (such as ergotaman-3', 6',18-trione,9,-10-dihydro-12'-hydroxy-2'-methyl-5'-(phenylmethyl)-,(5'(α))-, monomethanesulfonate, 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-[l-hydroxy-2-[(1-methyl-3-phenylpropyl) amino] ethyljsalicylamide monohydrochloride, e.g., Normodyne^®, Schering), bretylium tosylate (Benzenemethanaminium, 2-bromo-N-ethyl-N,N-dimethyl-, salt with 4-methylbenzenesulfonic acid (1 :1) <nplcit id="ncit0103" npl-type="c"><text>CAS RN 61-75-6</text></nplcit>), phentolamine mesylate (Phenol, 3-[[(4,5-dihydro-1H-imidazol-2-yl)methyl](4-methylphenyl)amino]-, monomethanesulfonate (salt) <nplcit id="ncit0104" npl-type="c"><text>CAS RN 65-28-1</text></nplcit>), solypertine tartrate (5H-1,3-Dioxolo[4,5-f]indole, 7-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-, (2R,3R)-2,3-dihydroxybutanedioate (1 :1) <nplcit id="ncit0105" npl-type="c"><text>CAS RN 5591-43-5</text></nplcit>), zolertine hydrochloride (Piperazine, 1-phenyl4-[2-(1H-tetrazol-5-yl)ethyl]-, monohydrochloride (8Cl, 9Cl) <nplcit id="ncit0106" npl-type="c"><text>CAS RN 7241-94-3</text></nplcit>) and the like; α adrenergic receptor blockers, such as alfuzosin (CAS RN: 81403-68-1), terazosin, urapidil, prazosin (Minipress^®), tamsulosin, bunazosin, trimazosin, doxazosin, naftopidil, indoramin, WHP 164, XENOlO, fenspiride hydrochloride (which may be prepared as disclosed in <patcit id="pcit0960" dnum="US3399192A"><text>US3399192</text></patcit>), proroxan (<nplcit id="ncit0107" npl-type="c"><text>CAS RN 33743-96-3</text></nplcit>), and labetalol hydrochloride and combinations thereof; α 2 agonists such as methyldopa, methyldopa HCL, lofexidine, tiamenidine, moxonidine, rilmenidine, guanobenz, and the like; aldosterone inhibitors, and the like; renin inhibitors including Aliskiren (SPPlOO; Novartis/Speedel); angiopoietin-2-binding agents such as those disclosed in <patcit id="pcit0961" dnum="WO03030833A"><text>WO03/030833</text></patcit>; anti-angina agents such as ranolazine (hydrochloride 1-Piperazineacetamide, N-(2,6- dimethylphenyl)-4-[2-hydroxy-3-(2-methoxyphenoxy)propyl]-, dihydrochloride <nplcit id="ncit0108" npl-type="c"><text>CAS RN 95635- 56-6</text></nplcit>), betaxolol hydrochloride (2-Propanol, l-[4-[2 (cyclopropylmethoxy)ethyl]phenoxy]-3-[(1-methylethyl)amino]-, hydrochloride <nplcit id="ncit0109" npl-type="c"><text>CAS RN 63659-19-8</text></nplcit>), butoprozine hydrochloride (Methanone, [4-[3(dibutylamino)propoxy]phenyl](2-ethyl-3-indolizinyl)-, monohydrochloride <nplcit id="ncit0110" npl-type="c"><text>CAS RN 62134-34-3</text></nplcit>), cinepazet maleatel-Piperazineacetic acid, 4-[l-oxo-3-(3,4,5- trimethoxyphenyl)-2-propenyl]-, ethyl ester, (2Z)-2-butenedioate (1 :1) <nplcit id="ncit0111" npl-type="c"><text>CAS RN 50679-07-7</text></nplcit>), tosifen (Benzenesulfonamide, 4-methyl-N-[[[(1S)-1-methyl-2-phenylethyl]amino]carbonyl]- <nplcit id="ncit0112" npl-type="c"><text>CAS RN 32295-184</text></nplcit>), verapamilhydrochloride (Benzeneacetonitrile, α-[3-[[2-(3,4-dimethoxyphenyl)ethyl]methylamino]propyl]-3 ,4-dimethoxy-α-( 1 -methylethyl)-, monohydrochloride <nplcit id="ncit0113" npl-type="c"><text>CAS RN 152-114</text></nplcit>), molsidomine (1,2,3-Oxadiazolium, 5-[(ethoxycarbonyl)amino]-3-(4-morpholinyl)-, inner salt <nplcit id="ncit0114" npl-type="c"><text>CAS RN 25717-80-0</text></nplcit>), and ranolazine hydrochloride (1 -Piperazineacetamide, N-(2,6-dimethylphenyl)4-[2-hydroxy-3-(2-methoxyphenoxy)propyl]-, dihydrochloride <nplcit id="ncit0115" npl-type="c"><text>CAS RN 95635-56-6</text></nplcit>); tosifen (Benzenesulfonamide, 4-methyl-N-[[[(lS)-1-methyl-2-phenylethyl]amino]carbonyl]- <nplcit id="ncit0116" npl-type="c"><text>CAS RN 32295-184</text></nplcit>); adrenergic stimulants such as guanfacine hydrochloride (such as N-amidino-2-(2,6-dichlorophenyl) acetamide hydrochloride, e.g., Tenex^® Tablets available from Robins); methyldopahydrochlorothiazide (such as levo-3-(3,4-dihydroxyphenyl)-2-methylalanine) combined with Hydrochlorothiazide (such as 6-chloro-3,4-dihydro-2H -1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide, e.g., the combination as, e.g., Aldoril^® Tablets available from Merck), methyldopachlorothiazide (such as 6-chloro-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-(2,6-dichlorophenylamino)-2-imidazoline hydrochloride, e.g., Catapres^®, Boehringer Ingelheim), clonidine (1H-Imidazol-2-amine, N-(2,6-dichlorophenyl)4,5-dihydro-<nplcit id="ncit0117" npl-type="c"><text>CAS RN 4205-90-7</text></nplcit>), I-lyzaar (Merck; a combination of losartan and hydrochlorothiazide), Co-Diovan (Novartis; a combination of valsartan and hydrochlorothiazide, Lotrel (Novartis; a combination of benazepril and amlodipine) and Caduet (Pfizer; a combination of amlodipine and atorvastatin), and those agents disclosed in <patcit id="pcit0962" dnum="US20030069221A"><text>US20030069221</text></patcit>.

1.2.2.11

Agents for the Treatment of Respiratory Disorders

0207The GCC agonist peptides described herein can be used in combination therapy with one or more of the following agents useful in the treatment of respiratory and other disorders including but not limited to: ( 1 ) β-agonists including but not limited to : albuterol (PRO VENTIL^® , S ALBUT AMOl^®) , VENTOLIN^®), bambuterol, bitoterol, clenbuterol, fenoterol, formoterol, isoetharine (BRONKOSOL^®, BRONKOMETER^®), metaproterenol (ALUPENT^®, METAPREL^®), pirbuterol (MAXAIR^®), reproterol, rimiterol, salmeterol, terbutaline (BRETHAIRE^®, BRETHINE^®, BRICANYL^®), adrenalin, isoproterenol (ISUPREL^®), epinephrine bitartrate (PRIMATENE^®), ephedrine, orciprenline, fenoterol and isoetharine; (2) steroids, including but not limited to beclomethasone, beclomethasone dipropionate, betamethasone, budesonide, bunedoside, butixocort, dexamethasone, flunisolide, fluocortin, fluticasone, hydrocortisone, methyl prednisone, mometasone, predonisolone, predonisone, tipred-aiie, tixocortal, triamcinolone, and triamcinolone acetonide; (3) β2-agonist-corticosteroid combinations [e.g., salmeterol-fluticasone (AD V AIR^®), formoterol-budesonid (S YMBICORT^®)] ; (4) leukotriene D4 receptor antagonists/leukotriene antagonists/LTD4 antagonists (i.e., any compound that is capable of blocking, inhibiting, reducing or otherwise interrupting the interaction between leukotrienes and the Cys LTI receptor) including but not limited to: zafhiukast, montelukast, montelukast sodium (SINGULAIR^®), pranlukast, iralukast, pobilukast, SKB-I06,203 and compounds described as having LTD4 antagonizing activity described in <patcit id="pcit0963" dnum="US5565473A"><text>U.S. Patent No. 5,565,473</text></patcit>; (5) 5 -lipoxygenase inhibitors and/or leukotriene biosynthesis inhibitors [e.g., zileuton and BAY1005 (CA registry 128253-31-6)]; (6) histamine H1 receptor antagonists/antihistamines (i.e., any compound that is capable of blocking, inhibiting, reducing or otherwise interrupting the interaction between histamine and its receptor) including but not limited to: astemizole, acrivastine, antazoline, azatadine, azelastine, astainizole, bromopheniramine, bromopheniramine maleate, carbinoxamine, carebastine, cetirizine, chlorpheniramine, chloropheniramine maleate, cimetidine clemastine, cyclizine, cyproheptadine, descarboethoxyloratadine, dexchlopheniramine, dimethindene, diphenhydramine, diphenylpyraline, doxylamine succinate, doxylamine, ebastine, efletirizine, epinastine, famotidine, fexofenadine, hydroxyzine, hydroxyzine, ketotifen, levocabastine, levocetirizine, levocetirizine, loratadine, meclizine, mepyramine, mequitazine, methdilazine, mianserin, mizolastine, noberastine, norastemizole, noraztemizole, phenindamine, pheniramine, picumast, promethazine, pynlamine, pyrilainine, ranitidine, temelastine, terfenadine, trimeprazine, tripelenamine, and triprolidine; (7) an anticholinergic including but not limited to: atropine, benztropine, biperiden, flutropium, hyoscyamine (e.g. Levsin^®; Levbid^®; Levsin/SL^®, Anaspaz^®, Levsinex timecaps^®, NuLev^®), ilutropium, ipratropium, ipratropium bromide, methscopolamine, oxybutinin, rispenzepine, scopolamine, and tiotropium; (8) an anti-tussive including but not limited to: dextromethorphan, codeine, and hydromorphone; (9) a decongestant including but not limited to: pseudoephedrine and phenylpropanolamine; (10) an expectorant including but not limited to: guafenesin, guaicolsulfate, terpin, ammonium chloride, glycerol guaicolate, and iodinated glycerol; (11) a bronchodilator including but not limited to: theophylline and aminophylline; (12) an anti-inflammatory including but not limited to: fluribiprofen, diclophenac, indomethacin, ketoprofen, S-ketroprophen, tenoxicam; (13) a PDE (phosphodiesterase) inhibitor including but not limited to those disclosed herein; (14) a recombinant humanized monoclonal antibody [e.g. xolair (also called omalizumab), rhuMab, and talizumab]; (15) a humanized lung surfactant including recombinant forms of surfactant proteins SP-B, SP-C or SP-D [e.g. SURTAXING, formerly known as dsc-104 (Discovery Laboratories)], (16) agents that inhibit epithelial sodium channels (ENaC) such as amiloride and related compounds; (17) antimicrobial agents used to treat pulmonary infections such as acyclovir, amikacin, amoxicillin, doxycycline, trimethoprin sulfamethoxazole, amphotericin B, azithromycin, clarithromycin, roxithromycin, clarithromycin, cephalosporins( ceffoxitin, cefmetazole etc), ciprofloxacin, ethambutol, gentimycin, ganciclovir, imipenem, isoniazid, itraconazole, penicillin, ribavirin, rifampin, rifabutin,amantaditic, rimantidine, streptomycin, tobramycin, and vancomycin; (18) agents that activate chloride secretion through Ca++ dependent chloride channels (such as purinergic receptor (P2Y(2) agonists); (19) agents that decrease sputum viscosity, such as human recombinant DNase 1, (Pulmozyme^®); (20) nonsteroidal anti-inflammatory agents (acemetacin, acetaminophen, acetyl salicylic acid, alclofenac, alminoprofen, apazone, aspirin, benoxaprofen, bezpiperylon, bucloxic acid, carprofen, clidanac, diclofenac, diclofenac, diflunisal, diflusinal, etodolac, fenbufen, fenbufen, fenclofenac, fenclozic acid, fenoprofen, fentiazac, feprazone, flufenamic acid, flufenisal, flufenisal, fluprofen, flurbiprofen, flurbiprofen, furofenac, ibufenac, ibuprofen, indomethacin, indomethacin, indoprofen, isoxepac, isoxicam, ketoprofen, ketoprofen, ketorolac, meclofenamic acid, meclofenamic acid, mefenamic acid, mefenamic acid, miroprofen, mofebutazone, nabumetone oxaprozin, naproxen, naproxen, niflumic acid, oxaprozin, oxpinac, oxyphenbutazone, phenacetin, phenylbutazone, phenylbutazone, piroxicam, piroxicam, pirprofen, pranoprofen, sudoxicam, tenoxican, sulfasalazine, sulindac, sulindac, suprofen, tiaprofenic acid, tiopinac, tioxaprofen, tolfenamic acid, tolmetin, tolmetin, zidometacin, zomepirac, and zomepirac); and (21) aerosolized antioxidant therapeutics such as S-Nitrosoglutathione.

1.2.2.12

Anti-Diabetic Agents

0208The GCC agonist peptides described herein can be used in therapeutic combination with one or more anti-diabetic agents, including but not limited to: PPARγ agonists such as glitazones (e.g., WAY-120,744, AD 5075, balaglitazone, ciglitazone, darglitazone (CP-86325, Pfizer), englitazone (CP-68722, Pfizer), isaglitazone (MIT/J&amp;J), MCC- 555 (Mitsibishi disclosed in <patcit id="pcit0964" dnum="US5594016A"><text>US5594016</text></patcit>), pioglitazone (such as such as Actos^™ pioglitazone; Takeda), rosiglitazone (Avandia^™;Smith Kline Beecham), rosiglitazone maleate, troglitazone (Rezulin^®, disclosed in <patcit id="pcit0965" dnum="US4572912A"><text>US4572912</text></patcit>), rivoglitazone (CS-Ol 1, Sankyo), GL-262570 (Glaxo Welcome), BRL49653 (disclosed in <patcit id="pcit0966" dnum="WO9805331A"><text>WO98/05331</text></patcit>), CLX-0921, 5-BTZD, GW-0207, LG- 100641, JJT-501 (JPNT/P&amp;U), L-895645 (Merck), R-119702 (Sankyo/Pfizer), NN-2344 (Dr. Reddy/NN), YM-440 (Yamanouchi), LY-300512, LY-519818, R483 (Roche), T131 (Tularik), and the like and compounds disclosed in <patcit id="pcit0967" dnum="US4687777A"><text>US4687777</text></patcit>, <patcit id="pcit0968" dnum="US5002953A"><text>US5002953</text></patcit>, <patcit id="pcit0969" dnum="US5741803A"><text>US5741803</text></patcit>, <patcit id="pcit0970" dnum="US5965584A"><text>US5965584</text></patcit>, <patcit id="pcit0971" dnum="US6150383A"><text>US6150383</text></patcit>, <patcit id="pcit0972" dnum="US6150384A"><text>US6150384</text></patcit>, <patcit id="pcit0973" dnum="US6166042A"><text>US6166042</text></patcit>, <patcit id="pcit0974" dnum="US6166043A"><text>US6166043</text></patcit>, <patcit id="pcit0975" dnum="US6172090B"><text>US6172090</text></patcit>, <patcit id="pcit0976" dnum="US6211205B"><text>US621 1205</text></patcit>, <patcit id="pcit0977" dnum="US6271243B"><text>US6271243</text></patcit>, <patcit id="pcit0978" dnum="US6288095B"><text>US6288095</text></patcit>, <patcit id="pcit0979" dnum="US6303640B"><text>US6303640</text></patcit>, <patcit id="pcit0980" dnum="US6329404B"><text>US6329404</text></patcit>, <patcit id="pcit0981" dnum="US5994554A"><text>US5994554</text></patcit>, <patcit id="pcit0982" dnum="WO9710813A"><text>WO97/10813</text></patcit>, <patcit id="pcit0983" dnum="WO9727857A"><text>WO97/27857</text></patcit>, <patcit id="pcit0984" dnum="WO9728115A"><text>WO97/28115</text></patcit>, <patcit id="pcit0985" dnum="WO9728137A"><text>WO97/28137</text></patcit>,<patcit id="pcit0986" dnum="WO9727847A"><text>WO97/27847</text></patcit>, <patcit id="pcit0987" dnum="WO0076488A"><text>WO00/76488</text></patcit>, <patcit id="pcit0988" dnum="WO03000685A"><text>WO03/000685</text></patcit>,<patcit id="pcit0989" dnum="WO03027112A"><text>WO03/027112</text></patcit>,<patcit id="pcit0990" dnum="WO03035602A"><text>WO03/035602</text></patcit>, <patcit id="pcit0991" dnum="WO03048130A"><text>WO03/048130</text></patcit>, <patcit id="pcit0992" dnum="WO03055867A"><text>WO03/055867</text></patcit>, and pharmaceutically acceptable salts thereof; biguanides such as metformin hydrochloride (N,N-dimethylimidodicarbonimidic diamide hydrochloride, such as Glucophage^™, Bristol-Myers Squibb); metformin hydrochloride with glyburide, such as Glucovance^™, Bristol-Myers Squibb); bufom-tin (Imidodicarbonimidic diamide, N-butyl-); etoformine (1-Butyl-2-ethylbiguanide, Schering A. G.); other metformin salt forms (including where the salt is chosen from the group of, acetate, benzoate, citrate, ftimarate, embonate, chlorophenoxyacetate, glycolate, palmoate, aspartate, methanesulphonate, maleate, parachlorophenoxyisobutyrate, formate, lactate, succinate, sulphate, tartrate, cyclohexanecarboxylate, hexanoate, octanoate, decanoate, hexadecanoate, octodecanoate, benzenesulphonate, trimethoxybenzoate, paratoluenesulphonate, adamantanecarboxylate, glycoxylate, glutamate, pyrrolidonecarboxylate, naphthalenesulphonate, 1-glucosephosphate, nitrate, sulphite, dithionate and phosphate), and phenformin; protein tyrosine phosphatase- IB (PTP-IB) inhibitors, such as A-401,674, KR 61639, OC- 060062, OC-83839, OC-297962, MC52445, MC52453, ISIS 113715, and those disclosed in <patcit id="pcit0993" dnum="WO99585521A"><text>WO99/585521</text></patcit>, <patcit id="pcit0994" dnum="WO9958518A"><text>WO99/58518</text></patcit>, <patcit id="pcit0995" dnum="WO9958522A"><text>WO99/58522</text></patcit>, <patcit id="pcit0996" dnum="WO9961435A"><text>WO99/61435</text></patcit>, <patcit id="pcit0997" dnum="WO03032916A"><text>WO03/032916</text></patcit>, <patcit id="pcit0998" dnum="WO03032982A"><text>WO03/032982</text></patcit>, <patcit id="pcit0999" dnum="WO03041729A"><text>WO03/041729</text></patcit>, <patcit id="pcit1000" dnum="WO03055883A"><text>WO03/055883</text></patcit>, <patcit id="pcit1001" dnum="WO0226707A"><text>WO02/26707</text></patcit>, <patcit id="pcit1002" dnum="WO0226743A"><text>WO02/26743</text></patcit>, <patcit id="pcit1003" dnum="JP2002114768B"><text>JP2002114768</text></patcit>, and pharmaceutically acceptable salts and esters thereof; sulfonylureas 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. disclosed in <patcit id="pcit1004" dnum="US4379785A"><text>US4379785</text></patcit>, such as Amaryl , Aventis), glipentide, glipizide (e.g. Glucotrol or Glucotrol XL Extended Release, Pfizer), gliquidone, glisolamide, glyburide/glibenclamide (e.g. Micronase or Glynase Prestab, Pharmacia &amp; Upjohn and Diabeta, Aventis), tolazamide (e.g. Tolinase), and tolbutamide (e.g. Orinase), and pharmaceutically acceptable salts and esters thereof; meglitinides such as repaglinide (e.g. Pranidin^®, Novo Nordisk), KAD1229 (PF/Kissei), and nateglinide (e.g. Starlix^®, Novartis), and pharmaceutically acceptable salts and esters thereof; α glucoside hydrolase inhibitors (or glucoside inhibitors) such as acarbose (e.g. Precose^™, Bayer disclosed in <patcit id="pcit1005" dnum="US4904769A"><text>US4904769</text></patcit>), miglitol (such as GLYSET^™, Pharmacia &amp; Upjohn disclosed in <patcit id="pcit1006" dnum="US4639436A"><text>US4639436</text></patcit>), camiglibose (Methyl 6-deoxy-6-[(2R,3R,4R,5S)-3,4,5-trihydroxy-2- (hydroxymethyl)piperidino]-alpha-D-glucopyranoside, Marion Merrell Dow), voglibose (Takeda), adiposine, emiglitate, pradimicin-Q, salbostatin, CKD-711, MDL-25,637, MDL- 73,945, and MOR 14, and the compounds disclosed in <patcit id="pcit1007" dnum="US4062950A"><text>US4062950</text></patcit>, <patcit id="pcit1008" dnum="US4174439A"><text>US4174439</text></patcit>, <patcit id="pcit1009" dnum="US4254256A"><text>US4254256</text></patcit>, <patcit id="pcit1010" dnum="US4701559A"><text>US4701559</text></patcit>, <patcit id="pcit1011" dnum="US4639436A"><text>US4639436</text></patcit>, <patcit id="pcit1012" dnum="US5192772A"><text>US5192772</text></patcit>, <patcit id="pcit1013" dnum="US4634765A"><text>US4634765</text></patcit>, <patcit id="pcit1014" dnum="US5157116A"><text>US5157116</text></patcit>, <patcit id="pcit1015" dnum="US5504078A"><text>US5504078</text></patcit>, <patcit id="pcit1016" dnum="US5091418A"><text>US5091418</text></patcit>, <patcit id="pcit1017" dnum="US5217877A"><text>US5217877</text></patcit>, <patcit id="pcit1018" dnum="US51091A"><text>US51091</text></patcit> and <patcit id="pcit1019" dnum="WOO147528A"><text>WOO 1/47528</text></patcit> (polyamines); α-amylase inhibitors such as tendamistat, trestatin, and Al -3688, and the compounds disclosed in <patcit id="pcit1020" dnum="US4451455A"><text>US4451455</text></patcit>, <patcit id="pcit1021" dnum="US4623714A"><text>US4623714</text></patcit>, and <patcit id="pcit1022" dnum="US4273765A"><text>US4273765</text></patcit>; SGLT2 inhibtors including those disclosed inUS6414126 and <patcit id="pcit1023" dnum="US6515117B"><text>US6515117</text></patcit>; an aP2 inhibitor such as disclosed in <patcit id="pcit1024" dnum="US6548529B"><text>US6548529</text></patcit>; insulin secreatagogues such as linogliride, A-4166, forskilin, dibutyrl cAMP, isobutylmethylxanthine (IBMX), and pharmaceutically acceptable salts and esters thereof; fatty acid oxidation inhibitors, such as clomoxir, and etomoxir, and pharmaceutically acceptable salts and esters thereof; A2 antagonists, such as midaglizole, isaglidole, deriglidole, idazoxan, caroxan, and fluparoxan, and pharmaceutically acceptable salts and esters thereof; insulin and related compounds (e.g. insulin mimetics) such as biota, LP-100, novarapid, insulin deternir, insulin lispro, insulin glargine, insulin zinc suspension (lente and ultralente), Lys-Pro insulin, GLP-I (1-36) amide, GLP-1 (73-7) (insulintropin, disclosed in <patcit id="pcit1025" dnum="US5614492A"><text>US5614492</text></patcit>), LY-315902 (Lilly), GLP-I (7-36)-NH2), AL-401 (Autoimmune), certain compositions as disclosed in <patcit id="pcit1026" dnum="US4579730A"><text>US4579730</text></patcit>, <patcit id="pcit1027" dnum="US4849405A"><text>US4849405</text></patcit>, <patcit id="pcit1028" dnum="US4963526A"><text>US4963526</text></patcit>, <patcit id="pcit1029" dnum="US5642868A"><text>US5642868</text></patcit>, <patcit id="pcit1030" dnum="US5763396A"><text>US5763396</text></patcit>, <patcit id="pcit1031" dnum="US5824638A"><text>US5824638</text></patcit>, <patcit id="pcit1032" dnum="US5843866A"><text>US5843866</text></patcit>, <patcit id="pcit1033" dnum="US6153632A"><text>US6153632</text></patcit>, <patcit id="pcit1034" dnum="US6191105B"><text>US6191105</text></patcit>, and <patcit id="pcit1035" dnum="WO8505029A"><text>WO 85/05029</text></patcit>, and primate, rodent, or rabbit insulin including biologically active variants thereof including allelic variants, more preferably human insulin available in recombinant form (sources of human insulin include pharmaceutically acceptable and sterile formulations such as those available from Eli Killy (Indianapolis, Ind. 46285) as Humulin^™ (human insulin rDNA origin), also see the THE PHYSICIAN'S DESK REFERENCE, 55.sup.th Ed. (2001) Medical Economics, Thomson Healthcare (disclosing other suitable human insulins); non-thiazolidinediones such as JT-501 and farglitazar (GW-2570/GI- 262579), and pharmaceutically acceptable salts and esters thereof; PPARα/γ dual agonists such as AR-HO39242 (Aztrazeneca), GW-409544 (Glaxo-Wellcome), BVT-142, CLX-0940, GW-1536, GW-1929, GW-2433, KRP-297 (Kyorin Merck; 5-[(2,4-Dioxo thiazolidinyl)methyl] methoxy-N-[[4-(trifluoromethyl)phenyl] methy]jbenzamide), L-796449, LR-90, MK-0767 (Merck/Kyorin/Banyu), SB 219994, muraglitazar (BMS), tesaglitzar (Astrazeneca), reglitazar (JTT-501) and those disclosed in <patcit id="pcit1036" dnum="WO9916758A"><text>WO99/16758</text></patcit>, <patcit id="pcit1037" dnum="WO9919313A"><text>WO99/19313</text></patcit>, <patcit id="pcit1038" dnum="WO9920614A"><text>WO99/20614</text></patcit>, <patcit id="pcit1039" dnum="WO9938850A"><text>WO99/38850</text></patcit>, <patcit id="pcit1040" dnum="WO0023415A"><text>WO00/23415</text></patcit>, <patcit id="pcit1041" dnum="WO0023417A"><text>WO00/23417</text></patcit>, <patcit id="pcit1042" dnum="WO0023445A"><text>WO00/23445</text></patcit>, <patcit id="pcit1043" dnum="WO0050414A"><text>WO00/50414</text></patcit>, <patcit id="pcit1044" dnum="WO0100579A"><text>WO01/00579</text></patcit>, <patcit id="pcit1045" dnum="WO0179150A"><text>WO01/79150</text></patcit>, <patcit id="pcit1046" dnum="WO02062799A"><text>WO02/062799</text></patcit>, <patcit id="pcit1047" dnum="WO03004458A"><text>WO03/004458</text></patcit>, <patcit id="pcit1048" dnum="WO03016265A"><text>WO03/016265</text></patcit>, <patcit id="pcit1049" dnum="WO03018010A"><text>WO03/018010</text></patcit>, <patcit id="pcit1050" dnum="WO03033481A"><text>WO03/033481</text></patcit>, <patcit id="pcit1051" dnum="WO03033450A"><text>WO03/033450</text></patcit>, <patcit id="pcit1052" dnum="WO03033453A"><text>WO03/033453</text></patcit>, <patcit id="pcit1053" dnum="WO03043985A"><text>WO03/043985</text></patcit>, <patcit id="pcit1054" dnum="WO031053976A"><text>WO 031053976</text></patcit>, <patcit id="pcit1055" dnum="US66459800"><text>U.S. application Ser. No. 09/664,598, filed Sep. 18, 2000</text></patcit>, <nplcit id="ncit0118" npl-type="s"><text>Murakami et al. Diabetes 47, 1841-1847 (1998</text></nplcit>), and pharmaceutically acceptable salts and esters thereof; other insulin sensitizing drugs; VPAC2 receptor agonists; GLK modulators, such as those disclosed in <patcit id="pcit1056" dnum="WO03015774A"><text>WO03/015774</text></patcit>; retinoid modulators such as those disclosed in <patcit id="pcit1057" dnum="WO03000249A"><text>WO03/000249</text></patcit>; GSK 3β/GSK 3 inhibitors such as 4-[2-(2-bromophenyl)-4-(4-fluorophenyl-1H-imidazol-5- yl]pyridine and those compounds disclosed in <patcit id="pcit1058" dnum="WO03024447A"><text>WO03/024447</text></patcit>, <patcit id="pcit1059" dnum="WO03037869A"><text>WO03/037869</text></patcit>, <patcit id="pcit1060" dnum="WO03037877A"><text>WO03/037877</text></patcit>, <patcit id="pcit1061" dnum="WO03037891A"><text>WO03/037891</text></patcit>, <patcit id="pcit1062" dnum="WO03068773A"><text>WO03/068773</text></patcit>, <patcit id="pcit1063" dnum="EP1295884A"><text>EP1295884</text></patcit>, <patcit id="pcit1064" dnum="EP1295885A"><text>EP1295885</text></patcit>, and the like; glycogen phosphorylase (HGLPa) inhibitors such as CP-368,296, CP-316,819, BAYR3401, and compounds disclosed in WOO 1/94300, <patcit id="pcit1065" dnum="WO0220530A"><text>WO02/20530</text></patcit>, <patcit id="pcit1066" dnum="WO03037864A"><text>WO03/037864</text></patcit>, and pharmaceutically acceptable salts or esters thereof; ATP consumption promotors such as those disclosed in <patcit id="pcit1067" dnum="WO030007990A"><text>WO03/0007990</text></patcit>; TRB3 inhibitors; vanilloid receptor ligands such as those disclosed in <patcit id="pcit1068" dnum="WO03049702A"><text>WO03/049702</text></patcit>; hypoglycemic agents such as those disclosed in <patcit id="pcit1069" dnum="WO03015781A"><text>WO03/015781</text></patcit> and <patcit id="pcit1070" dnum="WO03040114A"><text>WO03/040114</text></patcit>; glycogen synthase kinase 3 inhibitors such as those disclosed in <patcit id="pcit1071" dnum="WO03035663A"><text>WO03/035663</text></patcit> agents such as those disclosed in <patcit id="pcit1072" dnum="WO9951225A"><text>WO99/51225</text></patcit>, <patcit id="pcit1073" dnum="US20030134890A"><text>US20030134890</text></patcit>, <patcit id="pcit1074" dnum="WO0124786A"><text>WO01/24786</text></patcit>, and <patcit id="pcit1075" dnum="WO03059870A"><text>WO03/059870</text></patcit>; insulin-responsive DNA binding protein-1 (IRDBP-I) as disclosed in <patcit id="pcit1076" dnum="WO03057827A"><text>WO03/057827</text></patcit>, and the like; adenosine A2 antagonists such as those disclosed in <patcit id="pcit1077" dnum="WO03035639A"><text>WO03/035639</text></patcit>, <patcit id="pcit1078" dnum="WO03035640A"><text>WO03/035640</text></patcit>, and the like; PPARδ agonists such as GW 501516, GW 590735, and compounds disclosed in <patcit id="pcit1079" dnum="JP10237049B"><text>JP10237049</text></patcit> and <patcit id="pcit1080" dnum="WO0214291A"><text>WO02/14291</text></patcit>; dipeptidyl peptidase IV (DP-IV) inhibitors, such as isoleucine thiazolidide, NVP-DPP728A (1- [[[2-[(5-cyanopyridin-2-yl)amino]ethyl]amino]acetyl]-2-cyano-(S)-pyrrolidme, disclosed by <nplcit id="ncit0119" npl-type="s"><text>Hughes et al, Biochemistry, 38(36), 11597-11603, 1999</text></nplcit>), P32/98, NVP-LAF-237, P3298, TSL225 (tryptophyl-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid, disclosed by <nplcit id="ncit0120" npl-type="s"><text>Yamada et al, Bioorg. &amp; Med. Chem. Lett. 8 (1998) 1537-1540</text></nplcit>), valine pyrrolidide, TMC-2A/2B/2C, CD- 26 inhibitors, FE999011, P9310/K364, VIP 0177, DPP4, SDZ 274-444, 2-cyanopyrrolidides and 4-cyanopyrrolidides as disclosed by <nplcit id="ncit0121" npl-type="s"><text>Ashworth et al, Bioorg. &amp; Med. Chem. Lett., Vol. 6, No. 22, pp 1163-1166 and 2745-2748 (1996</text></nplcit>) ,and the compounds disclosed in <patcit id="pcit1081" dnum="US6395767B"><text>US6395767</text></patcit>, <patcit id="pcit1082" dnum="US6573287B"><text>US6573287</text></patcit>, <patcit id="pcit1083" dnum="US6395767B"><text>US6395767</text></patcit> (compounds disclosed include BMS-477118, BMS-471211 and BMS 538,305), <patcit id="pcit1084" dnum="WO9938501A"><text>WO99/38501</text></patcit>, <patcit id="pcit1085" dnum="WO9946272A"><text>WO99/46272</text></patcit>, <patcit id="pcit1086" dnum="WO9967279A"><text>WO99/67279</text></patcit>, <patcit id="pcit1087" dnum="WO9967278A"><text>WO99/67278</text></patcit>, <patcit id="pcit1088" dnum="WO9961431A"><text>WO99/61431</text></patcit><patcit id="pcit1089" dnum="WO03004498A"><text>WO03/004498</text></patcit>, <patcit id="pcit1090" dnum="WO03004496A"><text>WO03/004496</text></patcit>, <patcit id="pcit1091" dnum="EP1258476A"><text>EP1258476</text></patcit>, <patcit id="pcit1092" dnum="WO02083128A"><text>WO02/083128</text></patcit>, <patcit id="pcit1093" dnum="WO02062764A"><text>WO02/062764</text></patcit>, <patcit id="pcit1094" dnum="WO03000250A"><text>WO03/000250</text></patcit>, <patcit id="pcit1095" dnum="WO03002530A"><text>WO03/002530</text></patcit>, <patcit id="pcit1096" dnum="WO03002531A"><text>WO03/002531</text></patcit>, <patcit id="pcit1097" dnum="WO03002553A"><text>WO03/002553</text></patcit>, <patcit id="pcit1098" dnum="WO03002593A"><text>WO03/002593</text></patcit>, <patcit id="pcit1099" dnum="WO03000180A"><text>WO03/000180</text></patcit>, and <patcit id="pcit1100" dnum="WO03000181A"><text>WO03/000181</text></patcit>; GLP-I agonists such as exendin-3 and exendin-4 (including the 39 aa polypeptide synthetic exendin-4 called Exenatide^®), and compounds disclosed in <patcit id="pcit1101" dnum="US2003087821A"><text>US2003087821</text></patcit> and <patcit id="pcit1102" dnum="NZ504256"><text>NZ 504256</text></patcit>, and pharmaceutically acceptable salts and esters thereof; peptides including amlintide and Symlin^® (pramlintide acetate); and glycokinase activators such as those disclosed in <patcit id="pcit1103" dnum="US2002103199A"><text>US2002103199</text></patcit> (fused heteroaromatic compounds) and <patcit id="pcit1104" dnum="WOO248106A"><text>WOO2/48106</text></patcit> (isoindolin-1-one-substituted propionamide compounds).

0209All patents, patent applications, and publications mentioned herein are hereby incorporated by reference in their entireties. However, where a patent, patent application, or publication containing express definitions is incorporated by reference, those express definitions should be understood to apply to the incorporated patent, patent application, or publication in which they are found, and not to the remainder of the text of this application. In the case of conflict, the present specification, including definitions, will control. The references cited herein are not admitted to be prior art to the invention.

0210It is to be understood that while the invention has been described in conjunction with the preferred specific embodiments thereof, that the foregoing description is intended to illustrate and not limit the scope of the invention. It will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the scope of the invention, and further that other aspects, advantages and modifications will be apparent 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 WO 2012/118972)

Attachment Fmoc-AA-OH to 2-ClTrt resin

02112-ClTrt resin (10 g, substitution =1.0 mmol/g resin) was suspended in 100 mL of dichloromethane (DCM) for 5 minutes, and then drained. The esterification was performed using 1.5 equiv. of Fmoc-amino acid and 1.7 equiv. Diisopropylethylamine (DIEA) in 80 mL of DCM (with minimum quantity of dimethylformamide (DMF) to dissolve the amino acid completely) for 2 hours. The resulting resin was washed with 60 mL of DCM and endcapped with 60 mL of DIEA/methanol (1:9, v/v) solution for 30 minutes. The loaded resin was then washed with DCM (6 vol.) for 2 times, DMF (6 vol.) for 3 times and methyl t-butylether (MTBE) (6 vol.) for 3 times, and dried under high vacuum. The substitution of the Fmoc-protected resin was determined by Fmoc release assay. Finally, the Fmoc group was deprotected 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 vol.) for 2 times and the resin was washed and dried under high vacuum to give the final resin for peptide synthesis. The results of the experiments are listed in Table VIII below. <tables id="tabl0023" num="0023"><img file="EP4424697A2_D0037.tif" /></tables>

Synthesis of side-chain-protected fragments A and B

0212H-Gly-2-ClTrt resin or H-Leu-2-ClTrt resin was suspended in DMF (10 vol.) for 20 minutes, then drained. The resulting resin was washed with DMF (10 vol.) for 5 minutes. The chain assembly was conducted using the standard Fmoc chemistry. Generally, 1.5 equiv. of Fmoc amino acid and 1.5 equiv. of HOBt were dissolved in DMF (4.5 vol.), followed by addition of 1.5 equiv. of DIEA. Then, the resulting solution was cooled to below 5 °C with an ice water bath, and activated by addition of 1.5 equiv. of HBTU. DCM (1.5 vol.) was added to the resin, followed by 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 Kaiser Test. If Kaiser Test indicated the presence of unreacted amine after 2 hours, recoupling with the same protocol using 1.0 equiv. of Fmoc amino acid, 1.0 equiv. of HOBt and 1.0 equiv. of DIEA was required. Capping was generally achieved by acetylating the unreacted amine with a mixture of acetic anhydride/pyridine/DMF solution. The peptide sequence was assembled by repeating the above capping procedure with the corresponding Fmoc-amino acid derivatives in the sequence from C- to N-terminal. The coupling of Fmoc-Cys(Trt)-OH or Fmoc-Cys(Acm)-OH residue was achieved by using 2.0 equiv. of Fmoc-Cys(Trt)-OH or Fmoc-Cys(Acm)-OH, 2.0 of equiv. HOBt and 2.0 equiv. of DIC <i>in situ</i> activation in DCM/DMF protocol to minimize racemization of cysteine.

0213After completion of the synthetic step, the peptide resin was thoroughly washed with DMF (10 vol.), MTBE (10 vol.), DMF (10 vol. 3 times) and MTBE (10 vol. 3 times) and subsequently dried in a vacuum oven to a constant weight.

0214The side-chain-protected peptide was cleaved from the resin using 1%TFA/DCM (10 vol.) for 3 times, 5 minutes for each time and the cleavage fractions were collected onto pyridine each time (1:1 volume ratio to TFA in each cleavage fraction). The peptide resin was washed with DCM (7.5 vol.). The fractions were combined and concentrated under vacuum to 10% of the original volume, and the resulting solution was reconstituted with ethanol (3 vol.) and concentrated to 50% of the original volume. Finally, the peptide was precipitated out by addition of water (1 vol.). The solid was collected by vacuum filtration or centrifugation and washed with water twice. The product was dried in vacuum to a constant weight and subjected to HPLC and ES-MS analysis. The results of the experiments are presented in Table IX below. <tables id="tabl0024" num="0024"><img file="EP4424697A2_D0038.tif" /></tables><tables id="tabl0025" num="0025"><img file="EP4424697A2_D0039.tif" /></tables>

Example 2: Condensation of fragments of SP-304 in solution (as in WO 2012/118972)

Synthesis of fragment C: H-AA15-160tBu <b>(1-1)</b>

0215A solution of Fmoc-Cys(Acm)-OH (124.38 gm, 0.3 mol), H-Leu-OtBu.HCl (67.12 gm, 0.3 mol), and HOBt (40.54 gm, 0.3 mol) in DMF (600 mL) was cooled to -5 ⁰C. 2-[1H-Benzotriazole-1-yl]-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) (113.79 gm, 0.3 mol) was added and dissolved completely. DIEA (183.1 mL, 1.05 mol) was added dropwise over a period of 105 minutes at the same temperature with good stirring, keeping 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% H₃PO₄ (300 mL). The organic layer was separated and the aqueous layer was extracted with EtOAc (600 mL). The combined extracts were washed with 5% H₃PO₄ (2 times), H₂O (1 time), saturated NaHCO₃ (3 times), H₂O (2 times), and brine (2 times). The solution was dried over MgSO₄ anhydrous, filtered, and evaporated in vacuo to dryness. The product was recrystallized from petroleum ether/EtOAc (3:1) and dried: 166.75 gm (yield 95.0%, purity 99.0%).

0216Fmoc-Cys(Acm)-Leu-OtBu (166.75 gm, 0.277 mol) was dissolved in a 10% piperidine/DCM solution (810 mL) with stirring. The reaction was monitored with TLC. After the reaction was completed in 3 hours, the solvent and the volatile materials were removed using a rotavap. The oily material obtained was triturated with petroleum ether to remove the byproducts by decantation. The residue, a syrup, was taken up in EtOAc, and washed with a mixture solution of NaH₂PO₄/Na₂HPO₄ (pH=6), then saturated NaHCO₃, purified H₂O, and brine. The organic layer was dried over MgSO₄ anhydrous. Evaporation of the solvent and the volatile materials yielded an oily product H-Cys(Acm)-Leu-OtBu <b>(1-1)</b> (73.23 gm, yield: 73.1%, purity: 98.0%).

Synthesis of fragment B-C: HAA7-160tBu <b>(2-3)</b>

0217A solution of Fmoc-AA7-140H <b>(1')</b> (198.3 gm, 124.0 mmol), H-AA15-160tBu <b>(1-1)</b> (52.3 gm, 148.8 mmol) and Cl-HOBt (21.0 gm, 124.0 mmol) in DMF (2500 mL) was cooled to - 5 °C. HBTU (51.7 gm, 136.4 mmol) was added and dissolved completely. DIEA (54.1 mL, 310 mmol) was then added with stirring, keeping 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 warm up to 20-27 °C slowly and stirring was continued for one and a half hours. Then, the mixture was poured into precooled (10-20 °C) 0.5 N HCl aq. (20 L).The suspension was stored at 20-25 °C for 45 minutes. The solid was collected by filtering the suspension through a fritted-glass funnel of medium porosity and subsequent washing with 0.5 N HCl aq.(3500 mL, 2 times), purified water (3500 mL), saturated NaHCO₃ aq. (3500 mL, 2 times) and purified water (3500 mL, 2 times) and diethyl ether (2000 mL, 2 times). Finally, the wet, crude peptide FmocAA7-16OtBu was dried in a desiccator under high vacuum at room temperature to yield product 238.22 gm (purity, 85.27%, yield 98.9%).

0218To a solution of FmocAA7-16OtBu (234.88 gm, 120.9 mmol) in DMF (2350 mL) was added piperidine (123 mL, 1245.2mmol). The reaction mixture was stirred at 25°C for two and a half hours, and then the mixture was poured into n-hexane (20.0L). The resulting sticky precipitate was triturated with n-hexane (3500mL, 7 times). The sticky precipitate was dissolved in minimal amount of DMF (2000mL), and then poured into 0.5N HCl aq. (10 vol.). The solid was collected by filtration and washed with purified water (3 times), and then diethyl ether (3000mL, 3 times), dried in air overnight and then dried in vacuo to give product (<b>2-3</b>) (HAA7-16OtBu) (183.67gm, purity: 68.4%, yield%: 88.33%, ES-MS, MW: calculated=1721.2, found=1719.84).

Synthesis of the fully protected A-B-C: BocAA1-16OtBu (<b>3-3</b>)

0219A solution of HAA7-16OtBu (<b>2-3</b>) (183.67 gm, 106.71 mmol), BocAA1-6-OH (2') (157.65 gm, 106.71 mmol), and 6-Cl-HOBt (18.141 gm, 106.71 mmol) in DMF (3 L) were cooled to at -3 to 0 °C. HBTU (44.523 gm, 117.38 mmol) was added and dissolved completely. DIEA (55.8 mL, 320.13 mmol) was then added with stirring, keeping the pH of the mixture 6. Stirring was continued for 20 minutes at -5 to 0°C. The reaction mixture was allowed to warm up to 25 °C slowly and stirring was continued for 2.5 hours, followed by further addition of HBTU (4.048 gm, 10.671 mmol) and DIEA (2 mL). Stirring was continued for another 1.5 hours. The resulting mixture was poured into MeOH (15 L), and the precipitate was collected and washed with MeOH/DMF mixture (5:1, v/v) (2 times, 3 L), 0.1N HCl (3 L, 2 times), saturated NaHCO₃ (2 times), purified water (3 times), diethyl ether (2 times) and dried in vacuo to yield the product BocAA1-16OtBu (<b>3-3</b>) (278.0 gm, yield 82.0%. Note: the purity was determined after the deprotection).

Synthesis of the Partially Protected Linear SP-304: HAA1-16OH (<b>4-4</b>)

0220A mixture of TFA/TIS/EDT (8:1:1, 2400 mL) was cooled to (0-5°C) under nitrogen and Boc-AA1-16OtBu (<b>3-3</b>) (201 gm) was added in portions. The resulting suspension was stirred at 0-10°C for 30 min, then the reaction solution was allowed to warm up to 20-25°C with a water bath (10 minutes) and stirring was continued for additional 1hr and 50 min at the same temperature. The reaction mixture was poured into pre-cooled (10 °C) MTBE (18 L). Some heat was evolved during the addition of the peptide/TFA solution and the internal temperature went up to 5 °C. The resulting suspension was then stored 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 vacuum over P₂O₅, yielding 148.37 gm of off-white product, HAA1-16OH (<b>4-4</b>) (purity: 62.23%, ES-MS, MW: calculated=1828.07, found=1826.67).

Example 3: Oxidative cyclization and purification of SP-304 by polystyrenic absorbent resin (as in WO 2012/118972)

0221HAA1-16OH (<b>4-4</b>) (0.58 gm) 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 kept for an hour with monitoring the 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% HPLC, recovery 98.5% estimated from peak area). The resulting mixture was added 1% iodine/ACN dropwise 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. The iodine was quenched by addition of 0.5 M ascorbic acid aq. until the yellow disappeared. Then, the pH of the mixture was adjusted to 6-7 with 25% ammonia solution (51.0% HPLC, recovery 50% estimated from peak area).

0222The polystyrenic absorbent resin (D101) was packed to a 3(ID) × 9(L) CM column and well equilibrated with 6 column volume (CV) of ethanol, 4 CV of purified water, 2 CV of 5% HCl aq., 4 CV of purified water, 2 CV of 2% NaOH aq. and 4 CV of purified water at the flow rate of 3 CV/hour. The oxidized peptide solution was then loaded to the column at 2 CV/h. After loading, the column was washed with 2 CV purified water at 2 CV/h. The elution was conducted by applying 80% ethanol aq. to the column at 2 CV/h. The fractions with UV absorbents at 215 nm, was collected and combined (125 mL). The combined fractions were then evaporated in vacuum to 10% of the original volume and the suspension was precipitated with 125 mL of cold MTBE (10 vol). The solid was collected by filtration and dried in vacuum to yield the crude SP-304 (0.282 g, 55.0% HPLC).

Example 4: Oxidative cyclization and purification of SP-304 by RP-HPLC (as in WO 2012/118972)

0223Crude peptide (<b>4-4</b>), prepared as described in Example 2 was dissolved in 10% ACN aqueous to an approximate concentration of 1.25 g/L with continuous stirring via a mechanical stirrer. The pH of the peptide solution was adjusted to 8.5-9.0 with 20% ammonia aqueous and the resulting solution was stirred vigorously open to the atmosphere. Hydrogen peroxide (3%, 0.25 equiv.) was added and stirring was continued at room temperature for 60 minutes. The HPLC analysis showed complete consumption of the linear peptide. Then, the solution was acidified to pH 3-4 with 10% AcOH aqueous. The resulting solution was diluted to a concentration of about 1 g/L with purified water. Iodine (1.3% in ACN) was added in with vigorous stirring over a period of 10 minutes until the yellow color of the iodine persisted. At about half-hourly intervals samples were taken from the mixture and analyzed by RP-HPLC. The monocyclized peptide peak decreased gradually and a new peak (dicyclized peptide) emerged. Oxidation was complete when no monocyclized peptide peak left. The excess iodine was neutralized by a small amount of ascorbic acid. The resulting solution was loaded on a C₁₈ RP-HPLC column packed with Kromasil 100Å, 10µm silica gel. After the dicyclized peptide solution was loaded, a 3 column volume of a solution of 90% mobile phase A (1.0% TEA, 0.5%H₃PO₄ in H₂O, pH=7) and 10% mobile phase B (acetonitrile) was loaded to wash out lines. Then, gradient was operated from 10% B to 30% B in 80 minutes. Fractions were collected at recorded intervals when main peak began to elute. The purity of each fraction was monitored by analytical RP-HPLC. Fractions of purity ≤95% (not meeting the Main Pool criteria) were pooled accordingly and forward processed using the same buffer system and gradient elution parameters stated above. All fractions with purity ≥95% were pooled and stored at 2-8°C. The purified peptide solution was diluted in a 1:1 ratio with purified water and then loaded to the same RP-HPLC column. The counter-ion exchange was accomplished by washing the column with 2-3 column volumes of 0.5M ammonium acetate aqueous, followed by gradient elution from 90% C (0.2% AcOH aqueous solution) and10% mobile phase D (ACN) to 50% mobile phase C and 50% mobile phase D in 50 minutes. Fractions were collected at recorded intervals and monitored by analytical RP-HPLC. The fractions (≥95%) were collected and lyophilized to obtain final dry peptide, 68.0g (96.1% pure).

Example 5: Desalination and isolation of SP-304 after purification by RP-HPLC (as in WO 2012/118972)

0224After plecanatide was purified by RP-HPLC as described in Example 4, it was desalinated and isolated. Briefly, the purified plecanatide in ammonium acetate/acetonitrile/water buffer was loaded onto a column packed with polymeric absorbent (macroporous adsorption resin) and then eluted by a mixture of alcohol/water. Finally, the peptide alcohol solution was concentrated under reduced pressure, precipitated with an ether, e.g., diethyl ether or MTBE, and dried under vacuum to give the final product.

Resin (Polymeric Adsorbents) Screening

0225<b><i>Resin pre-treatment:</i></b> Polymeric adsorbents, DA201-C (from Jiangsu Suqing, China; crosslinked polystyrene; surface area 1200-1400 m²/g; average pore diameter: 3-4 nm; pore volume: 1.1-1.2 ml/g; bulk density: 0.68-0.75 g/ml; specific density: 1.03-1.1 g/ml; moisture: 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; crosslinked polystyrene; surface area ≥800 m²/g; average pore diameter: 6-8 nm; pore volume: 1.5-1.8 ml/g; bulk density: 0.65-0.70 g/ml; specific density: 1.02-1.07 g/ml; moisture: 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; crosslinked polystyrene; surface area 520-600 m²/g; average pore diameter: 25-30 nm; bulk density: 0.7-0.8 g/ml; moisture: 60-70%; particle size: 0.315∼1.25 mm ≧95%; uniformity coefficient: ≤1.6% ), and ADS-8 (from Nankai Hecheng, China; crosslinked polystyrene; surface area 450-500 m²/g; average pore diameter: 12-16 nm; bulk density: 0.65-0.75 g/ml; moisture: 60-70%; particle size: 0.315∼1.25 mm ≧95%; uniformity coefficient: ≤1.6%) were suspended in 4-6 volume of ethanol overnight. Decant or suction off the supernatant from the settled resin. Add 6-8 volume of deionized water and resuspend the resin with gentle overhead stirring. Again, decant or suction off the supernatant from the settled resin. Repeat the above water treatment and decantation steps until fines appearance is minimal.

0226<b><i>Column packing</i> and <i>regeneration:</i></b> Resuspend the pre-treated resins above with 1-2 volume of deionized water to form the resin slurry respectively by using gentle agitation. Pour the resin slurry slowly down the inside of the column to prevent air entrapment. After the resin slurry has been fully transferred to the column, rinse the inside of the column using a squirt bottle containing deionized water. Open the column outlet to from a settled resin bed (ID=4cm, H=10cm). Then the resin beds were washed successively at a flow rate of 3CV per hour by 4 CV of deionized water, 2CV of 5% HCl aq, 4CV of deionized water, 2CV of 2% NaOH aq and finally 4 CV of deionized water till the pH of the elute was around 7.

0227<b><i>Preparation of loading samples:</i></b> 2000mg of lyophilized plecanatide was dissolved in a mixture of 60mL of ACN and 150mL of 0.2% AcOH aq (the pH of the AcOH aq was adjusted to 4 with 10% ammonia aq.). After filtration with 1.2 µm Nylon membrane, the filtrate was diluted to 250 mL with 0.2% AcOH aq (pH4) and split into 4 parts (62.5 mL each) for loading.

0228<b><i>Loading the sample to the columns:</i></b> 62.5 mL of peptide solution above was loaded onto the above 4 columns at a flow rate of 2CV/h respectively. The loading elute was collected and tested by RP-HPLC to evaluate the absorbent capacity of each resin. The absorbent capacity results of each resin were demonstrated in Table X below. <tables id="tabl0026" num="0026"><img file="EP4424697A2_D0040.tif" /></tables>

0229<b><i>HPLC Method:</i></b> HPLC machine: Shimadzu LC-10AD vp; column: Kromasil, C18, 4.6×250mm ; mobile phase A: 0.1%TFA in water; mobile phase B: 0.1%TFA in ACN; detect at: 215nm; column temperature: 40°C; flow rate: 1.0mL/min; gradient: 25%B to 45% B in 30 min.

0230<b><i>Absorbent Capacity Calculation:</i></b> The absorbent capacity of each resin was demonstrated by the absorbent ratio of the peptide loaded onto each column, which was calculated by the quantity of the peptide absorbed in each resin column divided by the peptide quantity in each loading sample (500mg). The quantity of peptide absorbed in each column was calculated by the formula below:

0231<b>Quantity of peptide absorbed=</b>Quantity of peptide in the loading sample-Quantity of peptide in the loading elute= 500mg- 62.5mLx (1.6mg/mL x HPLC peak area of the eluate/HPLC peak area of the peptide standard solution)

0232<b><i>Washing the Column with Deionized Water:</i></b> The loaded columns above were then washed with 2CV of deionized water at 2CV/h to remove the salts. The washing eluates were collected and analyzed by RP-HPLC to determine the peptide quantity desorbed by water using the same method above. The desorbed peptide ratios of each resin were listed in Table XI below. <tables id="tabl0027" num="0027"><img file="EP4424697A2_D0041.tif" /></tables>

0233<b><i>Desorbing the Peptide with 90%Ethanol</i>/<i>Water:</i></b> After 2CV of water washes, the peptide absorbed in each column was then eluted by 1-2CV of 90% ethanol in water at 2 CV/h. The elution was collected and analyzed by RP-HPLC to determine the peptide quantity desorbed by 90% ethanol using the same method above. The desorbed peptide ratios of each resin were listed in Table XII below. <tables id="tabl0028" num="0028"><img file="EP4424697A2_D0042.tif" /></tables>

0234<b><i>Isolation of the Peptide from the Ethanol Solution:</i></b> The collected peptide/ethanol/water solution from each column was concentrated under reduced pressure, precipitated with MTBE, filtered and dried <i>in vacuo</i> to give the final product. The overall yield of the peptide processed by each column was demonstrated in Table XIII below. <tables id="tabl0029" num="0029"><img file="EP4424697A2_D0043.tif" /></tables><tables id="tabl0030" num="0030"><img file="EP4424697A2_D0044.tif" /></tables>

0235<b><i>Resin Screening Conclusion:</i></b> From the data above (Table X to Table XIII), the DA201-H resin presented the best absorbent capacity and the best desorption (by ethanol) performance for plecanatide among the resins in the experiment.

Desalination and Isolation Process Optimization

0236<b><i>Eluting Solvents Selection:</i></b> Isopropanol and ethanol are two commonly used solvents for eluting the peptide from the polymeric absorbents. Table XIV shows the amount of plecanatide that is able to be dissolved in aqueous ethanol or isopropanol solution depending on the v/v % isopropanol (or ethanol): water. <tables id="tabl0031" num="0031"><img file="EP4424697A2_D0045.tif" /></tables>

0237The water in the peptide/alcohol solution can be removed by azeotropic distillation. Table XV shows the property of the binary azeotropes of ethanol/water and isopropanol/water. <tables id="tabl0032" num="0032"><img file="EP4424697A2_D0046.tif" /></tables>

0238Degradation of plecanatide will occur during the long time storage of the peptide/alcohol/water solution and the concentration process. Table XVI demonstrates the stability data of plecanatide in 75% IPA/water solution and 90% EtOH/water at 23°C. <tables id="tabl0033" num="0033"><img file="EP4424697A2_D0047.tif" /></tables>

0239From the testing data obtained, plecanatide was fairly stable at 23 °C in alcohol/water solution within 6 hours.

0240Elution experiments were conducted using isopropanol/water mixtures with different v/v %. The desorption ratio and water content of the peptide eluates were listed in Table XVII below. <tables id="tabl0034" num="0034"><img file="EP4424697A2_D0048.tif" /></tables>

0241<b>75% <i>Isopropanol</i>/<i>water as eluting solution:</i></b> 500mg, of plecanatide (98.1 % pure) was dissolved in a mixture of 16 mL ACN and 49 mL of 0.2% AcOH aqueous (the pH of the 0.2%AcOH solution was adjusted to 4.0 by addition of 10% NH₄OH aq). After filtration by 1.2 µm nylon membrane, the peptide solution was loaded onto a column packed with DA201-H resin (ID=4cm, H=10cm, packed and pre-treated by the procedure mentioned previously) at 2CV/h. After loading, the column was washed with 2 CV deionized water at 2CV/h. Then, the peptide was eluted by 1.5CV of 75% IPA/water at 2CV/h. The elution was monitored by RP-HPLC. The eluate was collected (124mL, 98.3% pure). Karl Fisher analysis indicated water (65.9%) wt. %). Into a 1-neck, 500-mL round bottom flask was placed 124mL of 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 under reduced pressure (60 Pa) on a rotary evaporator and partially immersed in a 23 °C water bath. A whitish suspension was formed by feed-stripping approximately 622mL of isopropanol in 2 hours to about 1/3 of the initial volume of the solution (97.7% pure). Karl Fisher analysis indicated water (0.17% wt. %). To the concentrate was added 350 mL of pre-chilled diethyl ether, the solid was collected by centrifugation at 3500 rpm for 3 minutes and dried under vacuum to yield 333 mg of final product (yield 66.6%, 97.2% HPLC purity).

0242<b><i>95% Isopropanol</i>/<i>water as eluting solution I:</i></b> 500mg of plecanatide (98.1% pure) was dissolved in a mixture of 16 mL ACN and 49 mL of 0.2% AcOH aqueous (the pH of the 0.2%AcOH solution was adjusted to 4.0 by addition of 10% NH₄OH aq). After filtration by 1.2 µm nylon membrane, the peptide solution was loaded onto a column packed with DA201-H resin (ID=4cm, H=10cm, pre-treated by the procedure mentioned previously) at 2CV/h. After loading, the column was washed with 2 CV deionized water at 2CV/h. Then, the peptide was eluted by 1.5CV of 95% IPA/water at 2CV/h. The elution was monitored by RP-HPLC. The eluate was collected (117mL, 98.1% pure). Karl Fisher analysis indicated water (52% wt. %). Into a 1-neck, 500-mL round bottom flask was placed 117mL of peptide/IPA/water eluate collected above. The flask was then placed under reduced pressure (50 Pa) on a rotary evaporator and partially immersed in a 23 °C water bath. A whitish solid was formed by feed-stripping approximately 470mL of isopropanol in 130min (97.9%) pure). To the solid above was added 50 mL of pre-chilled diethyl ether to form a suspension and evaporated under reduced pressure (50Pa) on a rotary evaporator at 23°C to dryness. Yield was 430mg of final product (86%). HPLC purity was 97.9%.

0243<b><i>95% Isopropanol</i>/<i>water as eluting solution II:</i></b> 500 mg of plecanatide (98.1% pure) was dissolved in a mixture of 16 mL ACN and 49 mL of 0.2% AcOH aqueous (the pH of the 0.2%AcOH solution was adjusted to 4.0 by addition of 10% NH₄OH aq). After filtration by 1.2 µm nylon membrane, the peptide solution was loaded onto a column packed with DA201-H resin (ID=4cm, H=10cm, packed and pre-treated by the procedure mentioned previously) at 2CV/h. After loading, the column was washed with 2 CV deionized water at 2CV/h. Then, the peptide was eluted by 1.5CV of 95% IPA/water at 2CV/h. The elution was monitored by RP-HPLC. The eluate was collected (118mL, 98.2% pure). Karl Fisher analysis indicated water (53% wt. %). Into a 1-neck, 500-mL round bottom flask was placed 118mL of peptide/IPA/water eluate collected above. The flask was then placed under reduced pressure (50 Pa) on a rotary evaporator and partially immersed in a 23 °C water bath. A whitish suspension (~40mL) was formed by feed-stripping approximately 330mL of isopropanol in 100min (97.7% pure). To the suspension above was added 400 mL of pre-chilled diethyl ether to form a suspension. After standing at ambient temperature for 1hour, the solid was collected by centrifugation at 3500 rpm for 3 minutes, and dried under vacuum to yield 370 mg of final product. Yield was 74%. HPLC purity was 97.9%.

0244<b><i>95% Isopropanol</i>/<i>water as eluting solution III:</i></b> 10g of plecanatide was desalted and precipitated in a manner similar to that described above. Interestingly, the precipitation yield was improved to 93%, which is a significant increase in yield. HPLC purity after precipitation was 98.47%.

Example 6: Characterization of lyophilized SP-304 and precipitated SP-304 (as in WO 2012/118972)

0245The plecanatide purified by lyophilization as described in Example 4 and plecanatide purified by precipitation as described in Example 5 were analyzed to determine significant chemical impurity values such as the levels of acetamide, TFA, ammonium ion, and acetonitrile. The results are listed in the table below. <tables id="tabl0035" num="0035"><img file="EP4424697A2_D0049.tif" /></tables>

0246As demonstrated by results above, the precipitation process provided significantly reduced levels of undesirable process impurities.

0247The 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 densities, tap densities, particle size distribution, and shape.

0248<b>EQUIPMENT:</b> (1) Tap Density Tester Model TD-1020; (2) Sonic Sifter Separator Model L3P; (3) Optical Microscope LINITRON 2850.

METHODS:

0249<ol id="ol0003" compact="compact"><li>1) Bulk and Tap Density Measurements: Modified USP 1 Method <ol id="ol0004" compact="compact"><li>a. 100.0 mL graduate cylinder was used for lyophilized plecanatide</li><li>b. 10.0 mL graduated cylinder was used for precipitated plecanatide</li></ol></li><li>2) Particle Size Distribution Analysis <ol id="ol0005" compact="compact"><li>a. Screens used: 200, 140, 100, 60, 40 and 30 meshes.</li><li>b. Sample size: 2c; of lyophilized plecanatide and 6.4g of precipitated plecanatide</li></ol></li><li>3) Optical Microscopic Analysis: Particle Size and Shape <ol id="ol0006" compact="compact"><li>a. Dry powder was manually dispersed onto a microscopic plate</li><li>b. Magnification: 100 x</li><li>c. Under normal light condition (no polarized filters)</li></ol></li></ol>

RESULTS:

0250<ol id="ol0007"><li>(1) <i>Physical Appearance</i>: lyophilized plecanatide is a light, fluffy and white powder. Precipitated plecanatide is a slightly off-white powder.</li><li>(2) <i>Bulk</i> and <i>Tap Density</i>: Table XVIII provides bulk and tap density data for Plecanatide samples of both lyophilized and precipitated: <img file="EP4424697A2_D0050.tif" /> As seen from the data, the precipitated plecanatide is unexpectedly significantly denser than the lyophilized plecanatide. The precipitated plecanatide has less tendency of dust generation during processing, which affords the advantages of increased safety and reduced processing losses.</li><li>(3) <i>Particle Size Distribution:</i> Table XIX summarizes the particle size distribution analysis. <figref idref="f0001">Figure 1</figref> presents the data graphically. <img file="EP4424697A2_D0051.tif" /> As demonstrated by Table XIX and <figref idref="f0001">Fig. 1</figref>, the particle size distributions are different for both types of plecanatide. During analysis, it was observed that the precipitated plecanatide contained some larger particles, which could be broken up easily. It was also noticed that the lyophilized plecanatide appeared to be flaky and sticking onto top and bottom of sieves, whereas no sticking was observed for the precipitated plecanatide. It indicates a better processing property of the precipitated plecanatide.</li><li>(4) <i>Particle Size and Shape:</i><figref idref="f0002">Figures 2</figref> and <figref idref="f0003">3</figref> provide optical microscopic analysis of samples of lyophilized and precipitated plecanatide, respectively. As seen in <figref idref="f0002">Figure 2</figref>, the lyophilized plecanatide is in amorphous form and has irregular shapes of particles. They form physical aggregates with particles lying on top of each other. In <figref idref="f0003">Figure 3</figref>, the precipitated. plecanatide shows distinguishable individual particles. From the particle appearances and shapes, the precipitated plecanatide will have better flow property and therefore can facilitate solid processing during manufacturing.</li></ol>

0251The lyophilized and precipitated forms of plecanatide have shown distinguishable physical appearances and properties by density, particle size distribution and shape analyses. The precipitated form is more suitable for solid dosage form processing during manufacturing in terms of reducing dust generation, less sticking onto processing equipment, and potentially less processing losses.

0252The precipitated form is more suitable for processing solid dosage form during manufacture (e.g., a low-dose solid dosage form). The higher density of the precipitated material will reduce aerosol or "dust" losses during weighing, transferring, and blending. The different particle shape has been shown to reduce loss caused by sticking to screens or sieves. The higher density should improve content uniformity since the size and density of the drug particles more closely match those of the excipients.

Example 7: Low-dose formulation of precipitated SP-304 (as in WO 2012/118972)

0253The plecanatide purified by precipitation as described in Example 5 is processed further to make low-dose formulations as described below. <tables id="tabl0036" num="0036"><img file="EP4424697A2_D0052.tif" /></tables>

Blending:

0254Avicel PH 102 is screened through a 60 mesh screen. V-blenders (1 Qt, 4Qt, and 16 Qt) are then dusted by the screened Avicel PH 102. SP-304 is screened through a 200 mesh screen_ and loaded into the 1-Qt V-blender. Then, about 80 g Avicel PH 102 is added into the 1-Qt blender and the mixture is blended for 10 minutes at 25 rpm. The mixture is then transferred to the 4-Qt V-blender which is pre-dusted by the screened Avicel PH 102. The 1-Qt blender is rinsed with Avicel and the rinse material is transferred to the 4-Qt blender. The rinsing is repeated until all SP-304 is transferred to the 4-Qt blender. About 200g Avicel is added to the 4-Qt V-blender and the mixture is blended for 10 minutes. The resulting blend is then screened through a 60 mesh screen and then transferred into the pre-dusted 16-Qt blender (dusted with 1500g Avicel). The 4-Qt blender is rinsed with Avicel and the rinse material is transferred to the 16-Qt blender. The remaining Avicel is added to the 16-Qt blender and the mixture is blended for 10 minutes. The resulting blend is passed through Comil, rinsed with excess of Avicel, and then returned to the 16-Qt blender and is further blended for 5 minutes. Proper amount of magnesium stearate is weighed, screened through a 60 mesh screen, and added into the 16-Qt blender. The resulting mixture is blended for 2 minutes. The resulting mixture is then either packaged in capsules or compressed to form tablets.

Encapsulation

0255A MG2 Planeta capsule filler is set up. Average weight of the empty capsule shells is determined and target capsule fill weight was calculated (±5%). The blend from the above process is added into the hopper of the capsule filler and encapsulation is started. Run weight parameters are manually adjusted. Resulting capsules are then sorted according to the target fill weight.

Compression

0256A Fette tablet press is set up. Then the blend mixture is loaded into the powder hopper and tooling is installed. The weight of each tablet is set to be 100 mg±5% and hardness to be 4-6 Kp. The weight, hardness, and thickness of tablets are measured and recorded every 5 to 10 minutes. Friability measurement is also performed to ensure satisfactory product.

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

0257<b>Monocyclization of Linear Crude Peptide:</b> The disulfide bond between Cys⁴ and Cys¹² was first formed by H₂O₂ oxidation at 8.0-8.5 pH to form the monocyclized crude peptide. The linear crude peptide was slowly added and dissolving in 0.5% ammonium acetate in water buffer with pre-added H₂O₂ in a ratio of 100:9 gram of peptide to mL H₂O₂ to produce a final crude concentration of approximately 1.0 mg/mL. The solution pH was then adjusted to 8.5 with an NH₄OH solution while stirring in the open air. 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 monocyclized peptide, the oxidation reaction was stopped by adjusting the pH of peptide solution to 1.7-2 using a TFA solution. The peptide solution was then transferred to the next step for the formation of the dicyclized peptide.

0258<b>Dicyclization of Monocyclized Peptide:</b> The disulfide bond between the eys7 and Cys15 was formed by 3.0% (w/v) I₂ in acetonitrile solution. The disulfide bridge was created 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 quenched with a 0.1 M ascorbic acid in water solution. Upon completion of the reaction, the dicyclized peptide was adjusted to pH 6.5-7 using an NH₄OH solution and the material was prepared for primary purification.

0259<b>Primary Purification of Dicyclized Crude Peptide:</b> The dicyclized crude peptide solution resulting from the oxidation steps was then loaded onto a preparative RP-HPLC column packed with C₁₈ reverse phase resin which was operated by a preparative HPLC system. The peptide was eluted in a 1% triethylamine phosphate (TEAP) in water, pH7/ACN buffer system. HPLC Method 1 was used to ascertain the percent purity of fractions and pools obtained during the primary purification run.

0260<b>Recycle Pool(s) Purification of Dicyclized Peptide:</b> After primary purification, the fractions that required further purification were purified based on the purity of the fraction pools using a 1%TEAP in water, pH7/ACN or a 0.2% acetic acid in water/ ACN buffer system. The dicyclized crude peptide solution resulting from the oxidation steps was then loaded onto a preparative RP-HPLC column packed with C₁₈ reverse phase resin which was operated by a preparative HPLC system. The peptide was eluted in a 1%TEAP in water, pH7/ACN buffer system. HPLC Method 1 was used to ascertain the percent purity of fractions and pools obtained during the Recycle Purification run.

0261<b>Secondary Purification and Salt Exchange:</b> After recycle purification, the fractions that required further purification were purified based on the purity of the fraction pools using a 1%TEAP in water, pH7/ACN or a 0.2% acetic acid in water/ ACN buffer system. HPLC Method 1 was used to ascertain the percent purity of fractions and pools obtained during the Secondary Purification run. UPLC Method 1 was used to ascertain the percent purity of Main Pool obtained during all the Purification runs before moving to next step.

0262<b>Solvent Exchange:</b> Material meeting main pool criteria was loaded onto the preparative RP-HPLC column at the flange end in the reverse direction, washed with a 99:1 water to isopropanol solution from the reverse direction, and eluted with a 40:60 isopropanol : water solution in the forward direction. The collected peptide solution was tested by UPLC Method 1 to ascertain the purity, then the peptide solution was filtered, underwent rotary evaporation to remove excess isopropanol to below 5%, followed by sublot lyophilization for no less than 96 hours.

0263<b>Reconstitution and Final Lyophilization:</b> The sublot lyophilized dry peptide underwent reconstitution in water to form a homogenous lot. An amount of 0.5% (w/w) ammonium acetate to dry peptide was added to the solution and mixed until the ammonium acetate/peptide was dissolved. The material underwent analysis by UPLC Method 1 to verify the purity. The dissolved material was installed onto the tray lyophilizer and kept under vacuum for no less than 120 hours to comprise the final dry peptide material.

In Process Testing Methods

0264<b>HPLC Method 1:</b> The following RP-HPLC analytical method will be used to verify the HPLC percent purity of the primary and recycling purification steps or batch record indicated.

0265HPLC Parameters: <ul id="ul0003" list-style="none" compact="compact"><li>Column: Waters SunFire^™ C8, 3.5µm, 4.6 x 150mm or equivalent</li><li>Wavelength: 215 nm</li><li>Mobile Phase A: 0.02M TEAP in Water pH 6.5 (Analytical Buffer BA)</li><li>Mobile Phase B: 100% ACN</li><li>Injection Volume: 5-50 µL</li><li>Column Temp.: 40°C</li><li>Gradient Elution Parameters: <img file="EP4424697A2_D0053.tif" /></li></ul>

0266<b>HPLC Method 2:</b> The following RP-HPLC analytical method will be used to verify the HPLC percent purity of the linear crude peptide and to monitor the progress of the oxidative cyclization steps.

0267HPLC Parameters: <ul id="ul0004" list-style="none" compact="compact"><li>Column: Kromasil C18 5µ 100Å 4.5 x 250mm, or equivalent</li><li>Wavelength: 215 nm</li><li>Mobile Phase A: 0.02M TEAP in Water pH 6.5 (Analytical Buffer BA)</li><li>Mobile Phase B: 100% ACN</li><li>Injection Volume: 5-50 µL</li><li>Column Temp.: 40°C</li><li>Gradient Elution Parameters: <img file="EP4424697A2_D0054.tif" /><img file="EP4424697A2_D0055.tif" /></li></ul>

0268<b>UPLC Method 1:</b> The following RP-UPLC analytical method will be used to verify the Main Pool percent purity and final product peptide material before lyophilization .

0269UPLC Parameters: <ul id="ul0005" list-style="none" compact="compact"><li>Column: Waters Acquity BEH C₁₈, 1.7µm, 150 x 2.1mm, PIN 186002353 with a Waters Acquity column in-line filter unit, 0.2µm, P/N 205000343</li><li>Wavelength: 215 nm</li><li>Mobile Phase A: 48/ 52/ 0.16 - MeOH/ Water/ TFA</li><li>Injection Volume: 5µL</li><li>Column Temp.: 10°C</li><li>Isocratic Elution Parameters: <img file="EP4424697A2_D0056.tif" /></li></ul>

0270Two batches were prepared by the method described in the Example: lot#121026 IPA2 with a batch size of 50 g and lot#121026 B with a batch size of 930 g. As illustrated in <figref idref="f0004">Figure 4</figref>, lot#121026 IPA2 peptide had a UPLC chromatographic purity of ~98%. Other characteristics of the two batches are summarized in Table XX below, as compared to the batches prepared by the methods described in <patcit id="pcit1105" dnum="WO2012118972A"><text>WO 2012/118972</text></patcit>. <tables id="tabl0037" num="0037"><img file="EP4424697A2_D0057.tif" /></tables><tables id="tabl0038" num="0038"><img file="EP4424697A2_D0058.tif" /></tables><tables id="tabl0039" num="0039"><img file="EP4424697A2_D0059.tif" /></tables>

0271Other aspects of the invention are as follows: <ol id="ol0008"><li>1. A purified peptide comprising the GCC agonist sequence selected from the group consisting of SEQ ID NOs: 1, 9, and 104, wherein the purified peptide has the following characteristics: <ul id="ul0006" list-style="none" compact="compact"><li>a) has a bulk density of not greater than 0.1 g/mL;</li><li>b) contains less than 50 ppm acetamide;and</li><li>d) less than 0.3% alpha-Asp-9-plenacanatide (RRT 1.33).</li></ul></li><li>2. The purified peptide of paragraph 1, wherein the peptide is stable at 25 °C for at least three months;</li><li>3. The purified peptide of paragraph 1, wherein the peptide has a particle size distribution characterized by : <ol id="ol0009" compact="compact"><li>a) a D10 value of between about 2 -15 µm;</li><li>b) a D50 value of between about 15-50 µm; and</li><li>c) a D90 value of between about 40-80 µm</li></ol> when measured by light scattering with liquid dispersant.</li><li>4. The purified peptide of paragraph 1, wherein the peptide has a bulk density of not greater than 0.06 g/mL, not greater than 0.05 g/mL, or not greater than 0.04 g/mL,.</li><li>5. The purified peptide of paragraph 1 or 2, wherein the peptide has a chromatographic purity of no less than 95%, no less than 96%, or no less than 97%.</li><li>6. The purified peptide of any one of paragraphs 1-5, wherein the peptide is substantially free of water.</li><li>7. The purified peptide of paragraph 6, wherein the the water is not greater than 10%, not greater than 9%, not greater than 8%, not greater than 7%, not greater than 6% or not greater than 5%.</li><li>8. The purified peptide of any one of paragraphs 1-7, wherein the peptide is further substantially free of one or more impurities selected from acetonitrile, alcohols, ammonium, acetate, and TFA.</li><li>9. The purified peptide of paragraph 8, wherein the peptide contains less than 300 ppm acetonitrile.</li><li>10. The purified peptide of paragraph 8 or 9, wherein the peptide contains less than 0.1% TFA.</li><li>11. The purified peptide of any one of paragraphs 8-10, wherein the peptide contains less than 1000 ppm isopropanol.</li><li>12. The purified peptide of any one of paragraphs 8-11, wherein the peptide contains less than 600 ppm isopropanol.</li><li>13. The purified peptide of any one of paragraphs 8-12, wherein the peptide contains less than 0.25 % acetate.</li><li>14. The purified peptide of any one of paragraphs 1-13, wherein the peptide is further substantially free of topoisomers.</li><li>15. The purified peptide of any one of paragraphs 1-14, wherein the peptide is further substantially free of iso-Asp2-plecanatide (RTT 0.96-0.97)</li><li>16. A process of purifying a peptide comprising the GCC agonist sequence selected from the group consisting of SEQ ID NOs: 1-251, the process comprising: <ul id="ul0007" list-style="none" compact="compact"><li>providing a first peptide solution comprising a peptide comprising the GCC agonist sequence selected from the group consisting of SEQ ID NOs: 1-251, water, and acetonitrile;</li><li>loading a C18 or polymeric adsorbent column with the first peptide solution to adsorb the peptide onto the C19 or polymeric adsorbent column,</li><li>eluting the peptide off the C18 or polymeric adsorbent column with an alcohol aqueous solution to form a second peptide solution,</li><li>reducing the amount of alcohol in the second peptide solution, and</li><li>lyophilizing the second peptide solution such that a dry peptide is obtained.</li></ul></li><li>17. The process of paragraph 16, wherein the alcohol aqueous solution comprises isopropanol.</li><li>18. The process of paragraph 17, wherein the isopropanol content in the alcohol aqueous solution is about 40%.</li><li>19. The process of paragraph 16, wherein the first peptide solution further comprises acetamide.</li><li>20. The process of paragraph 16, wherein the first peptide solution further comprises 0.2% acetic acid or 1% triethylamine phosphate.</li><li>21. The process of paragraph 16, wherein the amount of alcohol in the second peptide solution is reduced by rotoevaporation.</li><li>22. The process of paragraph 16, wherein the alcohol in the second peptide solution is reduced to less than 5%.</li><li>23. The process of paragraph 22, wherein the alcohol in the second peptide solution is isopropanol.</li><li>24. The process of any one of paragraphs 17-23, further comprising dissolving the dry peptide in water to form a third peptide solution after lyophilization.</li><li>25. The process of paragraph 24, wherein the third peptide solution further comprises ammonium hydroxide or ammonium acetate.</li><li>26. The process of paragraph 24 or 25, further comprising lyophilizing the third peptide solution such that a purified peptide is obtained.</li><li>27. A purified peptide prepared by the process of any one of paragraphs 16-26.</li><li>28. The purified peptide of paragraph 27, wherein the peptide comprises the GCC agonist sequence selected from the group consisting of SEQ ID NOs: 1, 9, and 104.</li></ol>