EA040600B1 - GUANYLATE CYCLASE AGONISTS USED FOR THE TREATMENT OF OPIOID-INDUCED DYSFUNCTIONS - Google Patents

Description

Cross-reference to related applications

This application claims the benefit of US Provisional Application No. 61/889308, filed October 2013, which is incorporated herein by reference in its entirety for all purposes.

The field of technology to which the invention belongs

The present invention relates to the therapeutic use of guanylate cyclase C (GC-C) agonists for the treatment of opioid-induced dysfunctions such as opioid-induced stool disorder, opioid-induced esophageal upset, and opioid-induced respiratory depression. Agonists can be used either alone or simultaneously or sequentially with additional active agents to prevent, treat or attenuate one or more opioid-induced dysfunctions.

State of the art invention

Opioids are effective and widely used drugs for pain management in cancer and non-cancer patients. However, opioid use often has debilitating side effects such as opioid-induced dysfunction including respiratory depression, esophageal dysfunction, endocrine disruption, and opioid-induced stool disorder (OIBD) which includes opioid-induced constipation, dry mouth, nausea, vomiting, gastrostasis, bloating and pain in the abdomen.

There is a need for compositions and methods for treating and preventing the side effects of opioids.

The essence of the invention

The present invention provides a method for preventing, treating a condition, or alleviating a symptom of opioid-induced dysfunction by administering to a subject in need thereof a therapeutically effective amount of a composition that comprises a guanylate cyclase receptor agonist (GCRA) peptide. The composition may further comprise an opioid and/or a cGMP-dependent phosphodiesterase inhibitor. In one embodiment, the composition comprises a guanylate cyclase receptor (GCRA) agonist peptide and an opioid. In a further embodiment, the dosage form of the guanylate cyclase receptor agonist (GCRA) peptide and the opioid is presented as the same capsule or tablet. In a further embodiment, the opioid is selected from the group consisting of morphine, codeine, oxycodone, hydrocodone, dihydrocodeine, propoxyphene, fentanyl, tramadol, loperamide, butorphanol, or combinations thereof.

The compositions of the present invention may further contain a pharmaceutical carrier, excipient or diluent. In one embodiment, the composition comprises one or more guide materials selected from the group consisting of a pH dependent polymer, a swellable polymer, and a degradable composition. In a further embodiment, the formulation of the composition is optimized for delivery to the duodenum, jejunum, ileum, terminal ileum, or ascending colon. In another additional embodiment, the formulation of the composition is optimized for release throughout the colon. In another embodiment, the composition contains one or more pH dependent polymers that degrade in the pH range of 4.5 to 5.5, in the pH range of 5.5 to 6.5, or in the pH range of 6.5 to 7 ,5. In another additional embodiment, the composition is formulated in the form of a dosage form in such a way as to be released in a time-dependent manner.

The method described herein may further include the step of administering to the patient an effective dose of a cGMP-dependent phosphodiesterase inhibitor. For example, a cGMP-dependent phosphodiesterase inhibitor is sulindac sulfone, zaprinast, motapison, vardenafil, or sildenafil. The cGMP-dependent phosphodiesterase inhibitor may be administered either simultaneously or sequentially with the GCRA peptide or pharmaceutical composition thereof.

The present invention also provides a composition that contains an opioid and a GCRA peptide. The composition may further contain a pharmaceutical carrier, excipient or diluent.

Peptide-GCRA, which can be used in any method or any composition of the invention, has the amino acid sequence of any one of the table. 2-8. For example, the GCRA peptide is SEQ ID NO: 1 (SP-304), SEQ ID NO: 9 (SP-333), SP373 (SEQ ID NO: 104), SP364 (SEQ ID NO: 100), SP366 (SEQ ID NO: 102) or SED ID NO: 250.

Brief description of the drawings

Fig. 1 is a diagram showing the basic structure of uroguaniline and SP-333. Single letter abbreviations for amino acids are shown. SP-333 is similar to UG except for the substitution of aspartic acid (D) at position 3 from the N end of UG with glutamic acid (E). Additionally, the amino acids at both ends are replaced by their respective D-stereoisomers. Uroguaniline, like SP-333, has four cysteine (C) residues, allowing the formation of two intramolecular disulfide bonds. Substituted amino acids in SP-333 are shown in bold.

Fig. 2 is a diagram showing the mechanism of action of cyclic CMP and cyclic AMP in alleviating opioid-induced stool disorder (OBD).

Fig. 3A, 3B are a series of graphs showing that SP-333 apical treatment stimulated short circuit current in the T-84 cell monolayer and rat jejunum. Concentration dependent increase in chloride currents (measured as an increase in short circuit current) detectable after treatment of T-84 cells (A) and rat jejunal tissue (B) with SP-333. The concentrations of SP-333 that induced a half-maximal increase (EC50) in CI currents were comparable between cells and tissues; 1.69x10 ^-7 and 2.99x10 ^-7 M, respectively. Values are expressed as means ± standard error of the mean, n=4. T-84 cells and intact rat jejunum placed in a Ussing chamber were exposed to various concentrations of SP-333 in the apical chamber, and the maximum stimulation of short circuit current was recorded and plotted.

Fig. 4A, 4B are a series of graphs showing that SP-333 apical treatment stimulates short circuit current through activation of PKG-II and CFTR. Modulation of SP-333 (1 μm) stimulated apical short circuit current in T-84 cells (A), rat jejunum (B) in response to treatment with these inhibitors. T-84 cells and rat jejunum were placed in a Ussing chamber immersed apically and basolaterally in appropriate buffers as described in Example 1. To assess the role played by PKA and PKG-II and to identify the relative contributions of CFTR and CIC-2 into agonist-mediated release of Cl ions, experiments were performed in the presence and absence of kinase and CT channel inhibitors. The inhibitors used were CdCl ₂ (CIC-2 inhibitor; Sigma-Aldrich; St Louis, MO), CFTR _inh 172 (CFTR inhibitor; Santa Cruz Biotech; Dallas, Texas), KT5823 (PKG inhibitor; R&D Systems, Minneapolis, MN), mPKI (PKA inhibitor; EMD Millipore; Billerica, MA). The apical surface of T-84 cells (n=4 transwell/condition) and rat jejunal tissue (n=4-6 preparations/condition) were pretreated for 5 min with each inhibitor at the indicated concentration prior to addition of 1 μM SP-333. The peak values of the short circuit current obtained after the addition of SP-333 were recorded and calculated in terms of percentage values of relative activity ± standard error of the mean.

Fig. 5A, 5B are a series of graphs showing that morphine had no adverse effect on SP-333 stimulated short circuit current. T-84 cells (A) and rat jejunum (B) were pre-treated for 5 min with 5 μM morphine on the apical side. Subsequently, 1 μM SP-333 was added to the apical chamber and the stimulation of the short circuit current was recorded. The current recorded in the absence of opioids was taken as 100%. They built a graph of the relative short circuit current. Values are expressed as mean ± standard error of the mean, n=3 for T-84 cells and n=5-13 for rat jejunum.

Fig. 6A, 6B are a series of graphs showing that methadone had no adverse effect on SP-333 stimulated short circuit current. T-84 cells (A) and rat jejunum (B) were pre-treated for 5 min with 5 μM methadone on the apical side. Subsequently, 1 μM SP-333 was added to the apical chamber and the stimulation of the short circuit current was recorded. The current recorded in the absence of opioids was taken as 100%. They built a graph of the relative short circuit current. Values are expressed as mean ± standard error of the mean, n=3 for T-84 cells and n=5-13 for rat jejunum for each opioid.

Fig. 7A, 7B are a series of graphs showing that oral treatment with SP-333 increases gastrointestinal transit in rats. The left panel shows the dosing schedule for SP-333. Panel A is a bar graph showing that SP-333 increases gastrointestinal transit by 5.6%, 13%, and 23% at 0.05, 0.5, and 5 mg/kg, respectively, in opioid-naive animals. Panel B is a distribution plot showing the data for each individual animal.

Fig. 8A, 8B are a series of graphs showing that oral treatment with SP-333 improved gastrointestinal transit following morphine treatment. The indicated dose of morphine was administered to female Sprague-Dawley rats by intraperitoneal injection and gastrointestinal transit was assessed as described in Example 1. Morphine significantly reduced gastrointestinal transit in rats (A). Compared to medium controls, gastrointestinal transit after a single dose of morphine was slowed by 52%, 56%, and 56% at 0.25, 1.0, and 2.5 mg/kg opioid, respectively. The ability of SP-333 to reduce morphine-induced gastrointestinal transit delay was examined by administering 2.5 mg/kg morphine via intraperitoneal injection followed by indicated amounts of SP-333 via oral gavage feeding and assessing gastrointestinal transit as described in example 1. Morphine significantly reduced gastrointestinal transit (23% vs. 53% with medium treatment; p<0.0001) (B). A dose-dependent increase in gastrointestinal transit was observed after treatment with SP-333: 27%, 37%, 46%, and 48% at 0.5, 2.5, 5, and 50 mg/kg, respectively (P<0.002 at all doses except 0.5 mg /kg compared to the morphine-only dose group). A and B show the mean gastrointestinal transit ± standard error of the mean.

Fig. 9A, 9B are a series of graphs showing that oral treatment with SP-333 improved gastrointestinal transit after methadone treatment. A statistically significant decrease in gastrointestinal transit was observed in rats treated with 0.25, 2.5 and 5 mg/kg methadone (A). The delay in gastrointestinal transit observed with 2.5 mg/kg methadone was comparable to that observed with morphine at the same dose (49 and 56% vs. corresponding medium controls for methadone and morphine, respectively). SP-333 administration created a dose-dependent improvement in intestinal transit in methadone-treated rats: 40%, 39%, 44%, and 52% at 0.5, 2.5, 5, and 50 mg/kg, respectively (P<0.01 at all doses) . A and B show the mean gastrointestinal transit ± standard error of the mean.

Fig. 10A, 10B are a series of graphs showing that once daily treatment with SP-333 normalized gastrointestinal transit and restored fecal excretion after methadone treatment. Constipation was induced in rats by administering daily doses of 2.5, 5 and 7.5 mg/kg methadone for 7 days. Mean fecal excretion was recorded for 4 hours post-dose during the light period. A statistically significant decrease in fecal excretion was observed with all doses of methadone. To evaluate the ability of SP-333 to reduce methadone-induced constipation, rats were administered doses of 5 mg/kg of methadone once a day for 7 days. The dose of SP-333 was administered 10 minutes after the dose of methadone and fecal excretion was monitored as described above. SP-333 administered to methadone-treated animals at 2.5 mg/kg significantly improved methadone-induced constipation (B). A and B show the mean number of fecal lumps ± standard error of the mean per treatment group.

Fig. 11 is a graph showing that oral daily dosing of SP-333 reduces the delay in gastrointestinal transit caused by repeated dosing of methadone. The effect of repeated administration of methadone on gastrointestinal transit was monitored after rats received a once daily dose of 5 mg/kg methadone for 7 days followed by 2.5 mg/kg for the next week. On the day of testing, rats were given a dose of 2.5 mg/kg methadone followed by oral gavage of 2.5 and 5 mg/kg SP-333. Gastrointestinal transit was calculated as described in Example 1 and plotted as mean gastrointestinal transit ± standard error of the mean. Treatment with methadone resulted in a significant reduction in gastrointestinal transit. However, animals receiving a dose of SP-333 showed a statistically significant improvement in gastrointestinal transit delay.

Fig. 12 is a diagram demonstrating the mechanism of SP-333 mediated reduction in opioid-induced gastrointestinal transit delay. Morphine and methadone activate peripherally expressed opioid receptors, initiating downstream signals leading to lower cAMP levels due to inhibition of adenylyl cyclase, which affects the release of neurotransmitters. As a result, gastrointestinal motility, contractility, and fluid secretion are impaired, in combination with increased absorption of fluids and electrolytes from the intestinal lumen, leading to the manifestation of OIBD. By binding to the GC-C receptor on intestinal cells, SP-333 promotes the synthesis and accumulation of intracellular cGMP, which in turn binds and activates PKG-II directly or indirectly through inhibition of phosphodiesterase 3. PKG-II can phosphorylate and activate the CFTR channel , promoting the efflux of CI' ions, followed by a passive efflux of Na+ and water into the intestinal lumen, thus facilitating defecation. Although methadone can inhibit CIC-2, SP-333 mediated secretion of CI' from T-84 cells and rat jejunal tissue occurs via PKG-II and CFTR.

Detailed description

It should be understood that the singular forms are used throughout this application for convenience except where the context or unambiguous statement indicates otherwise, and the singular forms are intended to include the plural. Additionally, it should be understood that each journal article, patent, patent application, publication, and the like, which is mentioned in this document, is hereby incorporated by reference in its entirety and for all purposes. It should be understood that all numeric ranges include each and every numeric point within the numeric range and should be interpreted as listing each and every numeric point separately. The end values of all ranges directed to the same component or property are inclusive and can be independently combined.

Approximately contains all values that have substantially the same effect or provide substantially the same result as the standard value. Thus, the range covered by the term approximately will vary depending on the context in which the term is used, such as the parameter with which the standard value is associated. Thus, depending on the context, approximately can mean, for example, ±15, ±10, ±5, ±4, ±3, ±2, ±1%, or ±less than 1%. It is important that all standard value enumerations preceding the term approx. 3 040600 approx also assume that the standard value enumeration is compiled separately.

The present invention is based on the development of guanylate cyclase-C (GC-C) agonists. Agonists are analogues of uroguaniline, guaniline, lymphoguaniline and E. coli ST peptide.

Opioids are widely used to treat chronic pain, but their use is often associated with various side effects. Opioid-induced dysfunction includes respiratory depression, endocrine disruption, esophageal upset, and opioid-induced stool disorder. Opioid-induced dysfunction affects the gastrointestinal tract with severe constipation often seen in people for whom standard laxatives are not very effective. This side effect is due to a decrease in secretion of fluids, affecting gastrointestinal (GI) contractility and defecation (VM). Tab. 1 lists several non-limiting examples of the harmful effects of opioids and their clinical effect. The invention is based in part on the discovery of the use of GC-C agonists in the treatment of opioid-induced dysfunctions. The uroguaniline analog activates guanylate cyclase-C/cGMP (GC-C/cGMP) signaling to increase intestinal luminal fluid secretion to normalize GI and VM contractility and is used to treat opioid-induced constipation (OIC). This treatment completely restores the delay in gastrointestinal transit caused by morphine or methadone.

Table 1

Effects of opioids on the gastrointestinal tract

Scene Pharmacological Action Clinical effect Stomach Reduced contractility of the stomach Loss of appetite Decreased pylorus tone Nausea and vomiting Small intestine Decreased secretion of the pancreas and bile Delayed digestion Decreased peristalsis Delayed drug absorption Increased absorption of fluids Hard, dry stool Colon Decreased peristalsis Straining, incomplete emptying, bloating, bloating, constipation Increased non-propulsive contractions Cramping, abdominal cramps, pain Increased absorption of fluids Hard, dry stool Increased anal sphincter tone Incomplete emptying

Accordingly, the invention also provides a method for preventing, treating, or alleviating a symptom of opioid-induced dysfunction by administering to a patient, in need, a therapeutically effective amount of a GC-C agonist.

According to the invention, GC-C agonists include the amino acid sequences represented by formulas I-XX, as well as those amino acid sequences that are summarized below in table. 2-8. According to the invention, GC-C agonists are collectively referred to herein as GCRA peptides. In some embodiments, the GC-C agonist has the sequence SEQ ID NO: 1 (SP-304), SEQ ID NO: 9 (SP-333), SP373 (SEQ ID NO: 104), SP364 (SEQ ID NO: 100), SP366 (SEQ ID NO: 102) or SED ID NO: 250.

Opioids include, but are not limited to, alfentanil, anileridine, azimadolin, bremazocine, burprenorphine, butorphanol, codeine, deocine, diacetylmorphine (heroin), dihydrocodeine, diphenoxylate, ethylmorphine, fedotozine, fentanyl, funaltrexamine, hydrocodone, hydromorphone, levallorphan, levalmethadil acetate , levorphanol, loperamide, meperidine (pethidine), methadone, morphine, morphine-6-glucuronide, nalbuphine, nalorphine, nicomorphine, opium, oxycodone, oxymorphone, pantopone, pentazocine, propiram, propoxyphene, remifentanil, sufentanil, tilidine, trimebutine, and tramadol.

As used herein, opioid-induced dysfunction and the use of opioid side effects are used interchangeably and include such as, for example, gastrointestinal dysfunction (eg, inhibition of intestinal motility, constipation, contraction of GI sphincters, nausea, vomiting (vomiting mass)), biliary spasm, opioid colonic dysfunction, colic, dysphoria, pruritus, urinary retention, respiratory depression, pupillary constriction, cardiovascular effects, chest wall stiffness and cough suppression, reduced stress response, and associated immunosuppression with the use of narcotic analgesia or a combination thereof. Thus, the methods of the invention may be useful in terms of quality of life for patients tolerating the use of opioids, as well as to reduce the complications resulting from chronic constipation, such as hemorrhoids, appetite suppression, mucosal destruction, sepsis, cancer risk. colon and myocardial infarction.

In some embodiments, the provided GC-C agonists are used to administer to a subject undergoing acute opioid use. In some embodiments, the provided dosage forms are used for administration to patients suffering from postoperative gastrointestinal dysfunction, stool disorders, or respiratory depression.

In some embodiments, the provided dosage forms are also used for administration to subjects undergoing chronic opioid use (e.g., terminally ill patients receiving opioid therapy such as AIDS patients, cancer patients, patients with cardiovascular disease; subjects receiving chronic opioid therapy for pain management; subjects undergoing opioid therapy to maintain opioid withdrawal). In some embodiments, the patient is a patient using opioid therapy for long-term pain management. In some embodiments, the pain is non-cancerous pain (eg, back pain, neuropathic pain, pain associated with fibromyalgia, osteoarthritis). In some embodiments, the subject is a terminally ill patient. In other embodiments, the subject is a human undergoing maintenance therapy for opioid withdrawal.

In some embodiments, the dosage forms provided herein are administered to patients who have been selected for treatment with methadone or methylnaltrexone. In specific embodiments, the patient is selected on the basis of a patient having an increased risk of developing one or more of the pathological conditions set forth above. In yet another embodiment, the patient is selected based on the use of opioid therapy for pain management or based on the presence of one or more of the pathological conditions set forth herein. In some embodiments, the patient is constipated or has a history of constipation due to opioid therapy. In one embodiment, the constipated patient has not had a bowel movement in the previous three days. In one embodiment, the constipated patient has had fewer than three bowel movements in the previous week. In some embodiments, the constipated patient has had fewer than three drug-free bowel movements per week on average for the past four consecutive weeks, and one or more of the following:

(a) hard or lumpy stools;

(b) straining during bowel movements; and/or (c) feeling of incomplete emptying after a bowel movement.

The patient may use opioids intermittently or regularly.

In one embodiment, the patient who is selected has taken opioids as needed. In one embodiment, the selected patient has been on opioids for less than one week. In one embodiment, the selected patient has been on opioids for at least one week. In yet another embodiment, the selected patient has been on opioids for at least two weeks. In yet another embodiment, the selected patient has been on opioids for at least three weeks. In yet another embodiment, the selected patient has been on opioids for at least four weeks. In yet another embodiment, the selected patient has been on opioids for at least three months. In yet another embodiment, the selected patient has been on opioids for at least six months. In yet another embodiment, the selected patient has been on opioids for at least twelve months. In yet another embodiment, the patient that is selected has taken opioids for more than one year. In yet another embodiment, the selected patient has taken opioids at least every other day for at least two weeks. In one embodiment, the selected patient has received at least 7 doses of >25 mg oral morphine equivalents for at least 14 days. In one embodiment, the selected patient received a daily dose of >50 mg oral morphine equivalents for at least 14 days. In one embodiment, the selected patient suffers from constipation due to opioid therapy and has received a daily dose of >50 mg oral morphine equivalents for at least 14 days. In some embodiments, the patient received a daily dose of >50 mg oral morphine equivalents for at least 14 days and had fewer than three (3) drug-free bowel movements per week for an average of at least four consecutive weeks, that have been associated with one or more of the following:

(a) Bristol Stool Form Scale type 1 or 2 for at least 25% of non-medicated bowel movements;

(b) straining during at least 25% of defecations without medication; and/or (c) feeling of incomplete emptying after at least 25% of bowel movements without medication.

Non-drug defecation refers to defecation not associated with the use of a laxative within 24 hours before a bowel movement and/or (c) a feeling of incomplete emptying after a bowel movement.

In some embodiments, the provided dosage forms can be used in methods of preventing, inhibiting, reducing, delaying, reducing, or treating gastrointestinal dysfunction, including, but not limited to, irritable bowel syndrome, opioid-induced stool disorder, colitis, postoperative or postpartum ileus, nausea and/or vomiting, decreased gastric motility and emptying, inhibition of gastric motility and large and small propulsive movements of the small intestine, increased amplitude of non-propulsive segmental contractions, contraction of the sphincter of Oddi, increased tone of the anal sphincter, impaired reflex relaxation with rectal distention, decreased gastric secretion , bile, pancreas or small intestine, increased absorption of water from the contents of the colon, gastroesophageal reflux, gastric paresis, spasms, bloating, abdominal or epigastric pain and discomfort, constipation, idiopathic constipation, postoperative rational gastrointestinal dysfunction after abdominal surgery (eg, removal of the colon (eg, right-sided hemicolectomy, left-sided hemicolectomy, transverse colon resection, reoperation to restore intestinal continuity by anastomosis, low anterior rectal resection), hysterectomy) and suction delay orally administered drugs or nutrients.

GCRA peptides.

The GCRA peptides of the present invention are analogues of uroguaniline, guaniline, lymphoguaniline and ST peptides. The term peptide does not imply any particular length. In some embodiments, the GCRA peptide is less than 25 amino acids in length, such as less than or equal to 20, 15, 14, 13, 12, 11, 10, or 5 amino acids in length.

The GCRA peptides 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 inverse peptides. The term retro-inverse isomer refers to an isomer of a linear peptide in which the direction of the sequence is reversed and the chirality of each amino acid residue is reversed. See, for example, Jameson et al., Nature, 368, 744-746 (1994); Brady et al., Nature, 368, 692-693 (1994). The end result of combining D-enantiomers and reverse synthesis is that the positions of the carbonyl and amino groups in each amide bond are changed, while the position of the side chain groups in each alpha carbon atom is maintained. Unless specifically stated otherwise, it is contemplated that any given L-amino acid sequence of the invention can be converted to a D-retro-inverse peptide by reverse sequence synthesis for the corresponding native L-amino acid sequence. For example, peptide-GCRA contains the sequence defined by formulas I-XX, and the sequence listed in table. 2-8.

By inducing cGMP production is meant that the GCRA peptide induces the production of intracellular cGMP. Intracellular cGMP is measured using methods known in the art. For example, the GCRA peptides of the invention stimulate 5%, 10%, 20%, 30%, 40%, 50%, 75%, 90% or more of intracellular cGMP compared to naturally occurring GC-C agonists. In additional embodiments, the peptide-GCRA stimulates apoptosis, such as programmed cell death, or activates the cystic fibrosis transmembrane conductance regulator (CFTR).

As used herein, PEG3, 3 PEG is intended to refer to a polyethylene glycol that contains aminoethyloxy-ethyloxy-acetic acid (AeeA).

As used herein, the term amide is intended to mean that a terminal carboxylic acid has been replaced by an amide group, i. e. terminal COOH changed to CONH2.

As used herein, the term pyGlu refers to pyroglutamic acid.

As used in this document (for example, in formulas I-XX),

Xaa is any natural, synthetic amino acid or amino acid analogue;

Maa is cysteine (Cys), penicillamine (Pen), homocysteine, or 3-mercaptoproline;

Xaa _n1 is intended to refer to the amino acid sequence of any natural, artificial amino acid or amino acid analogue that is one, two or three residues in length;

Xaa _n2 is intended to refer to the amino acid sequence of any natural, artificial amino acid or amino acid analogue that is zero or one residue in length; And

- 6 040600

Xaa _n3 is intended to refer to the amino acid sequence of any natural, artificial amino acid, or amino acid analog that is zero, one, two, three, four, five, or six residues in length.

Additionally, any amino acid represented by Xaa may be an L-amino acid, a D-amino acid, a methylated amino acid, a fluorinated amino acid, or any combination thereof. Preferably the amino acids at the N-terminus, C-terminus, or both are D-amino acids. Optionally, any GCRA peptide represented by formulas I-XX may contain one or more polyethylene glycol residues at the N-terminus, C-terminus, or both. An exemplary polyethylene glycol contains aminoethyloxy-ethyloxy-acetic acid and its polymers.

Specific examples of GCC agonist peptides that can be used in the methods and dosage forms of the invention include a peptide selected from Table. 2-8.

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

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

In some embodiments, GCC agonist peptides include peptides having the formula III amino acid sequence, wherein at least one formula III amino acid is a D-amino acid or a methylated amino acid, and/or Maa is not a cysteine. Preferably the amino acid designated Xaa _n2 of formula III is a D-amino acid or a methylated amino acid. In some embodiments, the amino acid designated Xaan2 of Formula III is leucine, d-leucine, serine, or d-serine. Preferably, one or more of the amino acids represented by Xaa _n1 of formula III are D-amino acids or methylated amino acids. Preferably the amino acid at position Xaa ⁶ of formula III is leucine, serine or tyrosine.

In other embodiments, GCC agonist peptides include peptides having the formula IV amino acid sequence, wherein at least one formula IV amino acid is a D-amino acid or a methylated amino acid, and/or Maa is not a cysteine. Preferably Xaa _n2 of formula IV is a D-amino acid or a methylated amino acid. In some embodiments, the amino acid designated Xaan2 of Formula IV is leucine, d-leucine, serine, or d-serine. Preferably, one or more of the amino acids designated Xaa _n1 of formula IV are D-amino acids or methylated amino acids. Preferably the amino acid designated Xaa ⁶ of formula IV is leucine, serine or tyrosine.

In additional 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 (ie Xaa ¹⁶ ) of formula V is d-leucine or d-serine. Optionally, one or more amino acids at position 1-3 of Formula V are D-amino acids or methylated amino acids, or a combination of D-amino acids or methylated amino acids. For example, Asn ¹ , Asp ² or Glu ³ (or a combination thereof) of Formula V are D-amino acids or a methylated amino acid. Preferably the amino acid designated Xaa ⁶ of formula V is leucine, serine or tyrosine.

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

- 7 040600

In additional embodiments, GCRA peptides include peptides having the amino acid sequence of Formula X, XI, XII, XIII, XIV, XV, XVI, or XVII. Optionally, one or more of the amino acids of formulas X, XI, XII, XIII, XIV, XV, XVI or XVII are D-amino acids or methylated amino acids. Preferably the amino acid at the carboxyl end 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 carboxyl terminus of the peptides according to formulas X, XI, XII, XIII, XIV, XV, XVI or XVII is D-tyrosine.

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

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

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

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

In some embodiments, one or more amino acids of the GCRA peptides may be replaced with an artificial amino acid or a naturally occurring or artificial amino acid analogue. There are many amino acids beyond the standard 20 (Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val). Some exist in nature, others do not (see, for example, Hunt, The Non-Protein Amino Acids: In Chemistry and Biochemistry of the Amino Acids, Barrett, Chapman and Hall, 1985). For example, an aromatic amino acid can be replaced by 3,4-dihydroxy-b-phenylalanine, 3-iodo-b-tyrosine, triiodothyronine, L-thyroxine, phenylglycine (Phg) or nortyrosine (norTyr). Phg and norTyr and other amino acids including Phe and Tyr may be substituted with, for example, halogen, -CH ₃ , -OH, -CH ₂ NH ₃ , C(O)H, -CH ₂ CH ₃ , -CN, -CH ₂ CH ₂ CH ₃ , -Sh or another group. Any amino acid can be substituted with the D-form of the amino acid.

With regard to artificial amino acids or naturally occurring and artificial amino acid analogs, a number of substitutions in the polypeptide and agonists described herein are possible separately or in combination.

- 8 040600

For example, glutamine residues may be substituted with gamma-hydroxy-Glu or gamma-carboxyGlu. Tyrosine residues may be substituted with an alpha-substituted amino acid such as β-alphamethylphenylalanine or analogs such as 3-amino-Tyr; Tyr( _CH3 ); Tyr(PO ₃ (CH ₃ )2); Tyr(SO ₃ H); betacyclohexyl-Ala; beta-(1-cyclopentenyl)-Ala; beta-cyclopentyl-Ala; beta-cyclopropyl-Ala; betaquinolyl-Ala; beta-(2-thiazolyl)-Ala; beta-(triazol-1-yl)-Ala; beta-(2-pyridyl)-Ala; beta-(3-pyridyl)-Ala; amino Phe; fluoro-Phe; cyclohexyl-Gly; tBu-Gly; beta-(3-benzothienyl)-Ala; beta-(2-thienyl)-Ala; 5-methylTrp; and A-Methyl-Trp. Proline residues can be substituted with homopro (L-pipecolic acid); hydroxy-Pro; 3,4-dehydro-Pro; 4-Fluoro-Pro; or alpha-methyl-Pro or N(alpha)-C(alpha) cyclized amino acid analogs with the structure: n=0, 1, 2, 3. Alanine residues may be substituted with an 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 an artificial amino acid such as beta-fluoro-Ala. Alanine can also be substituted: n=0, 1, 2, 3. Glycine residues can be substituted with alpha-amino isobutyric acid (aib) or L/D-alpha-ethylalanine (L/D-isovaline).

Additional examples of artificial amino acids include an artificial analogue of alanine (eg, L-1-Nal or L-2-Nal); artificial analogue of tyrosine; artificial analogue of glutamine; artificial analogue of phenylalanine; artificial analogue of serine; artificial analogue of threonine; 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 photoactivated cross-linker; spin-labeled amino acid; a fluorescent amino acid; an amino acid with a new functional group; an amino acid that interacts covalently or non-covalently with another molecule; a metal-binding amino acid; an amino acid that is amidated at a site that is not naturally amidated, a metal-containing amino acid; radioactive amino acid; photolabile and/or photoisomerizable amino acid; an amino acid containing biotin or a biotin analog; a glycosylated or carbohydrate-modified amino acid; a ketogenic amino acid; amino acids containing polyethylene glycol or polyester; an amino acid with a substituted heavy element atom (eg, an amino acid containing deuterium, tritium, ¹³ C, ¹⁵ N, or ¹⁸ O); a chemically cleavable or photo-cleavable amino acid; an amino acid with an extended side chain; an amino acid containing a toxic group; a sugar-substituted amino acid such as a sugar-substituted serine and the like; a sugar-containing amino acid with an attached carbon; redox active amino acid; β-hydroxy containing acid; an amino acid containing an aminothioacid; α,α-disubstituted amino acid; β-amino acid; a cyclic amino acid that is not a proline; O-methyl-b-tyrosine; b-3-(2naphthyl)alanine; 3-methyl-phenylalanine; p-acetyl-b-phenylalanine; O-4-allyl-b-tyrosine; 4-propyl-b-tyrosine; tri-O-acetyl-GlcNAc β-serine; L-Dopa; fluorinated phenylalanine; isopropyl-b-phenylalanine; p-azido-b-phenylalanine; p-acyl-b-phenylalanine; p-benzoyl-b-phenylalanine; L-phosphoserine; phosphonoserine; phosphonothyrosine; p-iodo-phenylalanine; 4-fluorophenylglycine; p-bromophenylalanine; p-amino-b-phenylalanine; isopropyl-b-phenylalanine; b-3-(2-naphthyl)alanine; D-3-(2-naphthyl)alanine (dNal); amino-, isopropyl- or O-allyl-containing analogue of phenylalanine; dopa, O-methyl-b-tyrosine; glycosylated amino acid; p-(propargyloxy)phenylalanine; dimethyl-lysine; hydroxy-proline; mercaptopropionic acid; methyllysine; 3-nitro-tyrosine; norleucine; pyroglutamic acid; Z (carbobenoxyl); ε-acetyl-lysine; β-alanine; β-aspartic acid; β-cyclohexylalanine; an aminobenzoyl derivative; aminobutyric acid (Abu); citrulline; aminohexanoic acid (Ahx); aminoisobutyric acid (AIB); cyclohexylalanine; d-cyclohexylalanine; cyclohexylglycine; hydroxyproline; nitro-arginine; nitrophenylalanine; nitro-tyrosine; norvaline; octahydroindole carboxylate; ornithine (Orn); penicillamine (PEN); tetrahydroisoquinoline; diaminobutyric acid; diaminopimelic acid; pyroglutamic acid; homocysteine; homoserine; N-ε-dinitrophenyl-lysine; N-ε-methyl-lysine; Ns-dimethyllysine; N,N,N-ε-trimethyl-lysine; acetamidomethyl protected amino acids and pegylated amino acids. Additional examples of artificial amino acids and amino acid analogs can be found in US 20030108885, US 20030082575, US 20060019347 (paragraphs 410-418) and the references listed therein. The polypeptides of the invention may include additional modifications, including those described in US 20060019347, paragraph 589.

Nal as used herein refers to both b-1-naphthylalanine (b-1-Nal) and b-2-naphthylalanine (b-2-Nal).

In some embodiments, the amino acid may be replaced by a naturally occurring, non-essential amino acid, such as taurine.

Alternatively, GCRA peptides are cyclic peptides. Cyclic GCRA peptides are prepared using methods known in the art. For example, macrocyclization is often carried out through the formation of an amide bond between the N- and C-terminus of the peptide, between the side chain and the N- or C-terminus (for example, using K ₃ Fe(CN) ₆ at pH 8.5) (Samson et al ., Endocrinology,

- 9 040600

137:51825185 (1996)) or between two amino acid side chains such as cysteine. See, for example, DeGrado, Adv Protein Chem, 39:51-124 (1988). In some embodiments, the GCRA peptides of the present invention are bicyclic peptides. In various aspects, GCRA peptides are [4,12; 7.15] bicycles.

In some GCRA peptides, one or both elements of one or both pairs of Cys residues, which normally form a disulfide bond, can be replaced by homocysteine, penicillamine, 3-mercaptoproline (Kolodziej et al., 1996, Int J. Pept. Protein. Res. 48:274); β,β-dimethylcysteine (Hunt et al., 1993, Int. J. Pept. Protein. Res., 42:249) or diaminopropionic acid (Smith et al., 1978, J. Med. Chem., 2, 1: 117) to form alternative internal cross-links at conventional disulfide bond positions.

In addition, one or more disulfide bonds may be replaced by alternative covalent crosslinks, for example an amide bond (-CH ₂ CH(O)NHCH ₂ - or CH ₂ NHCH(O)CH ₂ -), an ester bond, a thioether bond, a lactam bridge, carbamoyl bond, urea bond, thiourea bond, phosphonate ester bond, alkyl bond (-CH2CH2CH2CH2-), alkenyl bond (-Ch ₂ Ch=CHCH ₂ -), ether bond (-CH2CH2OCH2- or -CH2OCH2CH2-), thioether bond (-CH2CH2SCH2- or -CH2SCH2CH2-), amine bond (-CH2CH2NHCH2- or -CH2NHCH2CH2-) or thioamide bond (-CH ₂ CH(S)HNHCH ₂ - or -CH ₂ NHCH(S)CH ₂ -). For example, Ledu et al. (Proc Nat'l Acad. Sci., 100:11263-78, 2003) describe methods for preparing lactam and amide crosslinks. Exemplary GCRA peptides that contain a lactam bridge include, for example, SP-370.

The GCRA peptides may have one or more conventional polypeptide linkages replaced with an alternative linkage. Such substitutions may increase the stability of the polypeptide. For example, replacing a polypeptide bond between the amino terminus of a residue with an aromatic residue (eg, Tyr, Phe, Tip) with an alternative bond may reduce cleavage of carboxy peptidases and may increase the half-life in the digestive tract. Bonds that can replace polypeptide bonds include retro-inverse bond (C(O)-NH instead of NH-C(O); reduced amide bond (NH-CH2); thiomethylene bond (S-CH2 or CH2-S); oxomethylene bond (O-CH2 or CH2-O); ethylene bond (CH2-CH2); thioamide bond (C(S)-NH); transolefin bond (CH=CH); fluorine substituted transolefin bond (CF=CH); ketomethylene bond (C(O)-CHR or CHR-C(O) in which R is H or CH ₃ ; and a fluoroketomethylene bond (C(O)-CFR or CFR-C(O) in which R is H or F or CH ₃ .

Peptides-GCRA can be modified using standard modifications. Modifications may occur at the amino (N-), carboxy (C-) terminus, within or in combination of any of the foregoing. In one aspect described herein, there may be more than one type of modification in a polypeptide. Modifications include, but are not limited to, acetylation, amidation, biotinylation, cinnamoylation, farnesylation, formylation, myristiolation, palmitoylation, phosphorylation (Ser, Tyr, or Thr), stearoylation, succinylation, sulfurylation, and cyclic formation (via disulfide bridges or cyclic amide formation) and modification with Cys3 or Cys5. The GCRA peptides described herein can also be modified with 2,4-dinitrophenyl (DNP), DNP-lysine, 7-amino-4-methyl-coumarin (AMC), fluorescein, NBD (7-nitrobenzene-2oxa -1,3-diazole), p-nitroanilide, rhodamine B, EDANS (5-((2-aminoethyl)amino)naphthalene-1sulfonic acid), dabcyl, dabsyl, dansyl, Texas red, FMOC, and Tamra (Tetramethylrhodamine) . The GCRA peptides described herein can also be conjugated to, for example, polyethylene glycol (PEG); alkyl groups (eg C1-C20 straight or branched alkyl groups); fatty acid radicals; combinations of PEG, alkyl groups and fatty acid radicals (see US Pat. No. 6,309,633; Soltero et al., 2001, Innovations in Pharmaceutical Technology, 106-110); BSA and KLH (Keyhole Limpet Hemocyanin). The addition of PEG and other polymers that can be used to modify the polypeptides of the invention is described in section IX of US 2006019347.

In one aspect, the invention provides an Aad-GCRA peptide as set forth in, for example, WO 20140151206, which is hereby incorporated by reference in its entirety for all purposes. Tab. 1 WO 20140151206 discloses examples of various alpha-aminoadipic acid derivatives of GCRA peptides. Aad-GCRA peptides are analogues of uroguaniline, guaniline, lymphoguaniline, and ST peptides. Specifically, these analogies contain α-aminoadipic acid (Ad), preferably at the 3 position from the N-terminus of each peptide or at the N-terminal position next to the first cysteine (Cys) residue. For example, an Aad-GCRA peptide contains sequences defined by formulas I, II, III, IV, V, VI, VII, VII, VIII, IX, XVIII, or XXI, which may contain α-aminoadipic acid.

In some embodiments, the Aad-GCRA peptide is Asn1-Asp ² -Aad ³ -Cys ⁴ Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ - Cys ¹⁵ -Leu ¹⁶ (SEQ ID NO: 251; SP-304-Aad), dAsn ^1-10 040600

Asp ² -Aad ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ^ll -Cys ¹² -Thr ^l3 -Gly ^l4 -Cys ^l5 -dLeu ¹⁶ (SEQ ID NO: 253 ;SP333-Aad); Pyglu'-Asp ² -Aad ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ^l0 -Ala ^ll -Cys ^l2 -Thr ^l3 -Gly ^l4 -Cys ^l5 -dLeu-AMIDE 16 ( SEQ ID NO: 254; SP-373-Aad), dAsn ^l -Asp ² -Aad ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Var-Ala-Cys ¹² -Thr ¹³ - Gly ¹⁴ Cys ¹⁵ -dSer ¹⁶ (SEQ ID NO: 255; SP-364-Aad); dAsn ¹ -Asp ² -Aad ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Var-Ala ¹¹ Cys ¹² -Thr ^l3 -Gly ¹⁴ -Cys ^l5 -dTyr ¹⁶ (SEQ ID NO: 256;SP-366-Aad); or Xaanl-Cys ⁴ -Xaa ⁵ -Xaa ⁶ -Xaa ⁷ -Xaa ⁸ -Xaa ⁹ Xaa ¹⁰ -Xaa ^ll -Cys ¹² -Xaa ¹³ -Xaa ^l4 -Xaa ^l5 -Xaan2 ¹⁶ (SEQ ID NO: 257).

Also included in the invention are peptides that are biologically or functionally equivalent to the peptides described herein. The term biological equivalent or functional equivalent means that the compositions of the present invention are capable of exhibiting some or all of the effects that modulate the production of cGMP.

Peptides-GCRAs may also contain derivatives of peptides-GCRAs, which are intended to include hybrid and modified forms of peptides-GCRAs in which some amino acids have been removed or replaced, and such modifications where one or more amino acids have been replaced with a modified amino acid or an unusual amino acid, and modifications such as glycosylation provided that the modified form retains the biological activity of the GCRA peptides. By retention of biological activity is meant that cGMP and/or apoptosis is induced by the GCRA peptide, although not necessarily at the same level of efficacy as the level of the naturally occurring GCRA peptide identified.

Preferred variants are those that have conservative amino acid substitutions made at one or more predicted non-essential amino acid residues. A conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (eg, threonine, valine, isoleucine), and aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted non-essential amino acid residue in the GCRA polypeptide is changed to another amino acid residue from the same side chain family. Alternatively, in yet another embodiment, all or part of the GCRA coding sequence can be randomly mutated, eg, by saturation mutagenesis, and the resulting mutants can be screened to identify mutants that retain activity.

table 2

Peptides-GCRA (SP-304 and derivatives)

Name Position of disulfide bonds Structure SEQ ID NO SP-304 C4:C12, C7:C15 Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Leu ¹⁶ 1 SP-326 SZ:S11, S6:S14 Asp ¹ -Glu ² -Cys ³ -Glu ⁴ -Leu ⁵ -Cys ⁶ -Val ⁷ -Asn ⁸ -Val ⁹ -Ala ¹⁰ -Cys ¹¹ -Thr ¹² -Gly ¹³ -Cys ¹⁴ -Leu ¹⁵ 2 SP-327 SZ:S11, S6:S14 Asp ¹ -Glu ² -Cys ³ -Glu ⁴ -Leu ⁵ -Cys ⁶ -Val ⁷ -Asn ⁸ -Val ⁹ -Ala ¹⁰ -Cys ¹¹ -Thr ¹² -Gly ¹³ -Cys ¹⁴ 3 SP-328 C2:C10, C5:C13 Glu ¹ -Cys ² -Glu ³ -Leu ⁴ -Cys ⁵ -Val ⁶ -Asn ⁷ -Val ⁸ -Ala ⁹ -Cys ¹⁰ -Thr ¹¹ -Gly ¹² -Cys ¹³ -Leu ¹⁴ 4 SP-329 C2:C10, C5:C13 Glu ¹ -Cys ² -Glu ³ -Leu ⁴ -Cys ⁵ -Val ⁶ -Asn ⁷ -Val ⁸ -Ala ⁹ -Cys ¹⁰ -Thr ¹¹ -Gly ¹² -Cys ¹³ 5 SP-330 C1:C9, C4:C12 Cys ¹ -Glu ² -Leu ³ -Cys ⁴ -Val ⁵ -Asn ⁶ -Val ⁷ -Ala ⁸ -Cys ⁹ -Thr ¹⁰ -Gly ¹¹ -Cys ¹² -Leu ¹³ 6 SP-331 C1:C9, C4:C12 Cys ¹ -Glu ² -Leu ³ -Cys ⁴ -Val ⁵ -Asn ⁶ -Val ⁷ -Ala ⁸ -Cys ⁹ -Thr ¹⁰ -Gly ¹¹ -Cys ¹² 7 SP332 C4:C12, C7:C15 Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ 8 SP-333 C4:C12, C7:C15 dAsn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ 9 SP-334 C4:C12,C7:C15 dAsn ¹ -dAsp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys 7 -Val ⁸ -Asn ⁹ -Val ^{10 -Ala 11} -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ 10 SP-335 C4:C12, C7:C15 dAsn ^{1 -dAsp 2} -dGlu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys 7 -Val 8 -Asn ^{9 -Val 10 -Ala 11 -Cys 12} -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ eleven SP-336 C4:C12, C7:C15 dAsn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Leu ¹⁶ 12 SP-337 C4:C12, C7:C15 dAsn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -dLeu ⁶ -Cys 7 -Val ⁸ -Asn ⁹ -Val ^{10 -Ala 11} -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ 13 SP-338 C4:C12, C7:C15 Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ 14 SP-342 C4:C12, C7:C15 PEG3-Asn ^{1 -Asp 2} -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys 7 -Val ^{8 -Asn} 9 -Val ^{10 -Ala 11} -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ - PEG3 15 SP-343 C4:C12, C7:C15 PEG3-dAsn ^{1 -Asp 2} -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys 7 -Val 8 -Asn ⁹ -Val ^{10 -Ala 11} -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ - PEG3 16 SP-344 C4:C12, C7:C15 PEG3-dAsn ^{1 -dAsp 2} -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys 7 -Val 8 -Asn ⁹ -Val ^{10 -Ala 11} -Cys ¹² -Thr ¹³ -Gly ¹⁴ Cys ¹⁵ dLeu ¹⁶ -PEG3 17 SP-347 C4:C12, C7:C15 dAsn ^{1 -Asp 2} -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val 8 -Asn 9 ^-Val 10 ^{-Ala 11} -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ -PEG3 18 SP-348 C4:C12, C7:C15 PEG3-Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys 7 ^-Val 8 -Asn ⁹ -Val ^{10 -Ala 11} -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ 19 SP-350 C4:C12, C7:C15 PEG3-dAsn ^{1 -Asp 2} -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys 7 -Val 8 -Asn ⁹ -Val ^{10 -Ala 11} -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ 20 SP-352 C4:C12, C7:C15 Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys 7 -Val ⁸ -Asn ⁹ -Val ^{10 -Ala 11} -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ -PEG3 21

- 11 040600

SP-358 C4:C12, C7:C15 PEG3-dAsn ^{1 -dAsp 2} -dGlu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val 8 -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ - PEG3 22 SP-359 C4:C12, C7:C15 PEG3-dAsn ^{1 -dAsp 2} -dGlu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys 7 -Val 8 -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ^{15 -dLeu 16} 23 SP-360 C4:C12, C7:C15 dAsn ^{1 -dAsp 2} -dGlu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys 7 -Val 8 -Asn 9 ^{-Val 10 -Ala 11 -Cys 12} -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ -PEG3 24 SP-361 C4:C12, C7:C15 dAsn ^{1 -dAsp 2} -Glu ^{3 -Cys 4} -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val 8 -Asn 9 -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ -PEG3 25 SP-362 C4:C12, C7:C15 PEG3-dAsn ^{1 -dAsp 2} -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys 7 -Val 8 -Asn ⁹ -Val ^{10 -Ala 11} -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ 26 SP-368 C4:C12, C7:C15 dAsn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dNal ¹⁶ 27 SP-369 C4:C12, C7:C15 dAsn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -AIB ⁸ -Asn ⁹ -AIB ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ 28 SP-370 S4:S12, 7:15 dAsn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -LeuVAsp[JIaKTaMHoii] ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Om ¹⁵ -dLeu ¹ 29 SP-371 C4:C12,C7:C15 dAsn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Tyr ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ thirty SP-372 C4:C12,C7:C15 dAsn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Ser ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ 31 N.I. C4:C12, C7:C15 PEG3-dAsn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ^{5 -Tyr 6} -Cys ⁷ -Val 8 -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ - PEG3 32 N2 C4:C12, C7:C15 PEG3-dAsn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ^{5 -Tyr 6} -Cys 7 -Val 8 -Asn ⁹ -Val ^{10 -Ala 11} -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ 33 N3 C4:C12,C7:C15 dAsn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ^{5 -Tyr 6} -Cys ⁷ -Val ⁸ -Asn 9 -Val 10 ^{-Ala 11} -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ -PEG3 34 N4 C4:C12,C7:C15 PEG3- ^{dAsn 1} -Asp ² -Glu ³ -Cys ⁴ -Glu ^{5 -Ser 6} -Cys 7 -Val 8 -Asn 9 -Val ^{10 -Ala 11} -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ - PEG3 35 N5 C4:C12, C7:C15 PEG3- ^{dAsn 1 -Asp 2} -Glu ³ -Cys ⁴ -Glu ⁵ -Ser ⁶ -Cys 7 -Val 8 -Asn 9 -Val ^{10 -Ala 11} -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ 36 N6 C4:C12, C7:C15 dAsn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Ser ⁶ -Cys ^{7 -Val 8} -Asn 9 -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dLeu ¹⁶ -PEG3 37 N7 C4:C12,C7:C15 Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Ser ¹⁶ 38 N8 C4:C12,C7:C15 PEG3-Asn ^{1 -Asp 2} -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys 7 -Val 8 -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ^{13 -Gly 14} -Cys ¹⁵ -Ser ¹⁶ - PEG3 39 N9 C4:C12,C7:C15 PEG3-Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Ser ¹⁶ 40 N10 C4:C12,C7:C15 Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Ser ¹⁶ -PEG3 41 Nil C4:C12,C7:C15 PEG3- ^{Asn 1 -Asp 2} -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys 7 -Val 8 -Asn 9 -Val ^{10 -Ala 11} -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dSer ¹⁶ - PEG3 42 N12 C4:C12,C7:C15 PEG3-Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dSer ¹⁶ 43 N13 C4:C12, C7:C15 Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dSer ¹⁶ -PEG3 44 Formula I C4:C12, C7:C15 Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Xaa ⁵ -Xaa ⁶ -Cys ⁷ -Xaa ⁸ -Xaa 9 -Xaa ¹⁰ -Xaa ¹¹ -Cys ¹² -Xaa ^{13 -Xaa 14} -Cys ¹⁵ -Xaa ¹⁶ 45 Formula II C4:C12, C7:C15 Xaa _n i-Cys ⁴ -Xaa ⁵ -Xaa ⁶ -Cys ⁷ -Xaa ⁸ -Xaa ⁹ -Xaa ¹⁰ -Xaa ¹¹ -Cys ¹² -Xaa ¹³ -Xaa ¹⁴ -Cys ¹⁵ -Xaa _n2 ¹⁶ 46 Formula III 4:12,7:15 Xaa _nl -Maa ⁴ -Glu ⁵ -Xaa ⁶ -Maa ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Maa ¹² -Thr ¹³ -Gly ¹⁴ -Maa ¹⁵ -Xaa _n2 47 Formula IV 4:12,7:15 Xaa _n i-Maa ⁴ -Xaa ⁵ -Xaa ⁶ -Maa ⁷ -Xaa ⁸ -Xaa ⁹ -Xaa ¹⁰ -Xaa ⁿ -Maa ¹² -Xaa ¹³ -Xaa ¹⁴ -Maa ¹⁵ -Xaa _n2 48 Formula V C4:C12,C7:C15 Asn ¹ -Asp ² -Asp ³ -Cys ⁴ -Xaa ⁵ -Xaa ⁶ -Cys 7 -Xaa ⁸ -Asn ⁹ -Xaa ¹⁰ -Xaa ¹¹ -Cys ¹² -Xaa ^{13 -Xaa 14} -Cys ¹⁵ -Xaa ¹⁶ 49 Formula VI C4:C12, C7:C15 dAsn ¹ -Glu ² -Glu ³ -Cys ⁴ -Xaa ⁵ -Xaa ⁶ -Cys 7 -X3 ⁸ -Asn 9 -Xaa ^{10 -Xaa 11 -Cys 12} -Xaa ¹³ -Xaa ¹⁴ -Cys ^{15 -d} -Xaa ¹⁶ 50 Formula Vll-a C4:C12, C7:C15 dAsn ¹ -dGlu ² -Asp ³ -Cys ⁴ -Xaa ^{5 -Xaa 6} -Cys 7 -Xaa 8 -Asn ⁹ -Xaa ¹⁰ -Xaa ¹¹ -Cys ^{12 -Xaa 13 -Xaa 14} -Cys ^{15 -d} -Xaa ¹⁶ 51 Formula VII-b C4:C12, C7:C15 dAsn ¹ -dAsp ² -Glu ³ -Cys ⁴ -Xaa ^{5 -Xaa 6} -Cys 7 -Xaa 8 -Asn ⁹ -Xaa ¹⁰ -Xaa ¹¹ -Cys ¹² -Xaa ¹³ -Xaa ¹⁴ -Cys ^{15 -d} -Xaa ¹⁶ 52 Formula VIII C4:C12,C7:C15 dAsn ^{1 -dAsp 2} -dGlu ³ -Cys ⁴ -Xaa ⁵ -Xaa ⁶ -Cys 7 -Xaa 8 -Tyr ⁹ -Xaa ¹⁰ -Xaa ¹¹ -Cys ^{12 -Xaa} 13 -Xaa ¹⁴ -Cys ^{15 -d} -Xaa ¹⁶ 53 Formula IX C4:C12, C7:C15 dAsn ¹ -dGlu ² -dGlu ³ -Cys ⁴ -Xaa ⁵ -Xaa ⁶ -Cys 7 -Xaa 8 -Tyr ⁹ -Xaa ¹⁰ -Xaa ¹¹ -Cys ^{12 -Xaa} 13 -Xaa ¹⁴ -Cys ^{15 -d} - ^{Xaa 16} 54 Formula XXI C4:C12, C7:C15 Xaa _nl -Cys ⁴ -Xaa ⁵ -Xaa ⁶ -Xaa ⁷ -Xaa ⁸ -Xaa ⁹ -Xaa ¹⁰ -Xaa ¹¹ -Cys ¹² -Xaa ¹³ -Xaa ¹⁴ -Xaa ¹⁵ -Xaa _n2 ¹⁶ 250

- 12 040600

Linaclotide and derivatives

Table 3

Name Position of disulfide bonds Structure SEQIDNO: SP-339 (linaclotide) C1:C6, C2:C10, C5:C13 Cys ¹ -Cys ² -Glu3-Tyr ⁴ -Cys ⁵ -Cys ⁶ -Asn ⁷ -Pro ⁸ -Ala ⁹ -Cys ¹⁰ -Thr ¹¹ -Gly ¹² -Cys ¹³ -Tyr ¹⁴ 55 SP-340 C1:C6, C2:C10, C5:C13 Cys ¹ -Cys ² -Glu ³ -Tyr ⁴ -Cys ⁵ -Cys ⁶ -Asn ⁷ -Pro ⁸ -Ala ⁹ -Cys ¹⁰ -Thr ¹¹ -Gly ¹² -Cys ¹³ 56 SP-349 C1:C6, C2:C10, C5:C13 PEG3-Cys ¹ -Cys ² -Glu ³ -Tyr ⁴ -Cys ⁵ -Cys ⁶ -Asn ⁷ -Pro ⁸ -Ala ⁹ -Cys ¹⁰ -Thr ¹¹ -Gly ¹² -Cys ¹³ -Tyr ¹⁴ -PEG3 57 SP-353 SZ:S8, S4:S12, S7:S15 Asn ¹ -Phe ² -Cys ³ -Cys ⁴ -Glu ⁵ -Ser ^{6 -Cys 7} -Cys 8 -Asn ⁹ -Pro ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Tyr ¹⁶ 58 SP-354 SZ:S8, S4:S12, S7:S15 Asn ¹ -Phe 2'Cys ³ -Cys ⁴ -Glu ⁵ -Phe ^{6 -Cys 7} -Cys ⁸ -Asn ⁹ -Pro ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Tyr ¹⁶ 59 SP-355 C1:C6, C2:C10, C5:C13 Cys ¹ -Cys ² -Glu ³ -Tyr ⁴ -Cys ⁵ -Cys ⁶ -Asn ⁷ -Pro ⁸ -Ala ⁹ -Cys ¹⁰ -Thr ¹¹ -Gly ¹² -Cys ¹³ -dTyr ¹⁴ 60 SP-357 C1:C6, C2:C10, C5:C13 PEG3-Cys ¹ -Cys ² -Glu ³ -Tyr ⁴ -Cys ⁵ -Cys ⁶ -Asn ⁷ -Pro ⁸ -Ala ⁹ -Cys ¹⁰ -Thr ¹¹ -Gly ¹² -Cys ¹³ -Tyr ¹⁴ 61 SP-374 SZ:S8, S4:S12, S7:S15 Asn ¹ -Phe ² -Cys ³ -Cys ⁴ -Glu ^{5 -Thr 6 -Cys 7} -Cys ⁸ -Asn ⁹ -Pro ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Tyr ¹⁶ 62 SP-375 SZ:S8, S4:S12, S7:S15 Asn ¹ -Phe ² -Cys ³ -Cys ⁴ -Glu ⁵ -Ser ⁶ -Cys ⁷ -Cys ⁸ -Asn ⁹ -Pro ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dTyr ¹⁶ 63 SP-376 SZ:S8, S4:S12, S7:S15 dAsn ¹ -Phe ² -Cys ³ -Cys ⁴ -Glu ⁵ -Ser ⁶ -Cys ⁷ -Cys ⁸ -Asn ⁹ -Pro ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Tyr ¹⁶ 64 SP-377 SZ:S8, S4:S12, S7:S15 dAsn ¹ -Phe ² -Cys ³ -Cys ⁴ -Glu ⁵ -Ser ⁶ -Cys ⁷ -Cys ⁸ -Asn ⁹ -Pro ^{10 -Ala 11} -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dTyr ¹⁶ 65 SP-378 SZ:S8, S4:S12, S7:S15 Asn ¹ -Phe ² -Cys ³ -Cys ⁴ -Glu ⁵ -Thr ⁶ -Cys ⁷ -Cys ⁸ -Asn ⁹ -Pro ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dTyr ¹⁶ 66 SP-379 SZ:S8, S4:S12, S7:S15 dAsn ¹ -Phe ² -Cys ³ -Cys ⁴ -Glu ⁵ -Thr ⁶ -Cys ⁷ -Cys ⁸ -Asn ⁹ -Pro ^{10 -Ala 11} -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Tyr ¹⁶ 67 SP-380 SZ:S8, S4:S12, S7:S15 dAsn ¹ -Phe ² -Cys ³ -Cys ⁴ -Glu ⁵ -Thr ^{6 -Cys} 7 -Cys ⁸ -Asn ⁹ -Pro ^{10 -Ala 11} -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dTyr ¹⁶ 68 SP-381 NW:S8, S4:S12, S7:15 Asn ¹ -Phe ² -Cys ³ -Cys ⁴ -Glu ⁵ -Phe ⁶ -Cys ⁷ -Cys ⁸ -Asn ⁹ -Pro ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dTyr ¹⁶ 69 SP-382 NW:S8, S4:S12, S7:15 dAsn ¹ -Phe ² -Cys ³ -Cys ⁴ -Glu ⁵ -Phe ⁶ -Cys ⁷ -Cys ⁸ -Asn ⁹ -Pro ^{10 -Ala 11} -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Tyr ¹⁶ 70 SP-383 NW:S8, S4:S12, S7:15 dAsn ¹ -Phe ² -Cys ³ -Cys ⁴ -Glu ⁵ -Phe ⁶ -Cys ⁷ -Cys ⁸ -Asn ⁹ -Pro ^{10 -Ala 11} -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dTyr ¹⁶ 71 SP384 C1:C6, C2:C10, C5:C13 Cys ¹ -Cys ² -Glu ³ -Tyr ⁴ -Cys ⁵ -Cys ⁶ -Asn ⁷ -Pro ⁸ -Ala ⁹ -Cys ¹⁰ -Thr ¹¹ -Gly ¹² -Cys ¹³ -Tyr ¹⁴ -PEG3 72 N14 C1:C6, C2:C10, C5:C13 PEG3-Cys ¹ -Cys ² -Glu ³ -Tyr ⁴ -Cys ⁵ -Cys ⁶ -Asn ⁷ -Pro ⁸ -Ala ⁹ -Cys ¹⁰ -Thr ¹¹ -Gly ¹² -Cys ¹³ -PEG3 73 N15 C1:C6, C2:C10, C5:C13 PEG3-Cys ¹ -Cys ² -Glu ³ -Tyr ⁴ -Cys ⁵ -Cys ⁶ -Asn ⁷ -Pro ⁸ -Ala ⁹ -Cys ¹⁰ -Thr ¹¹ -Gly ¹² -Cys ¹³ 74 N16 C1:C6, C2:C10, C5:C13 Cys ¹ -Cys ² -Glu ³ -Tyr ⁴ -Cys ⁵ -Cys ⁶ -Asn ⁷ -Pro ⁸ -Ala ⁹ -Cys ¹⁰ -Thr ¹¹ -Gly ¹² -Cys ¹³ -PEG3 75 N17 SZ:S8, S4:S12, S7:S15 PEG3- ^{Asn 1} -Phe ² -Cys ³ -Cys ⁴ -Glu ⁵ -Ser ⁶ -Cys ^{7 -Cys 8 -Asn 9 -Pro 10} -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ - Tyr ¹⁶ - PEG3 76 N18 SZ:S8, S4:S12, S7:S15 PEG3-Asn ¹ -Phe ² -Cys ³ -Cys ⁴ -Glu ⁵ -Ser ⁶ -Cys ⁷ -Cys ^{8 -Asn 9 -Pro 10} -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ - Tyr ¹⁶ 77 N19 SZ:S8, S4:S12, S7:S15 Asn ¹ -Phe ² -Cys ³ -Cys ⁴ -Glu ⁵ -Ser ⁶ -Cys ⁷ -Cys ⁸ -Asn ⁹ -Pro ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Tyr ¹⁶ -PEG3 78 N20 SZ:S8, S4:S12, S7:S15 PEG3-Asn ¹ -Phe ^{2 -Cys 3} -Cys ⁴ -Glu ⁵ -Phe ⁶ -Cys ^{7 -Cys 8 -Asn 9 -Pro 10} -Ala ¹¹ -Cys ^{12 -Thr 13 -Gly 14} -Cys ¹⁵ - Tyr ¹⁶ - PEG3 79 N21 SZ:S8, S4:S12, S7:S15 PEG3-Asn ¹ -Phe ² -Cys ³ -Cys ⁴ -Glu ⁵ -Phe ⁶ -Cys ⁷ -Cys ^{8 -Asn 9 -Pro 10 -Ala 11} -Cys 12 -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ - Tyr ¹⁶ 80 N22 SZ:S8, S4:S12, S7:S15 Asn ¹ -Phe ² -Cys ³ -Cys ⁴ -Glu ⁵ -Phe ⁶ -Cys ⁷ -Cys ⁸ -Asn ⁹ -Pro ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Tyr ¹⁶ -PEG3 81 N23 SZ:S8, S4:S12, S7:S15 PEG3-Asn ¹ -Phe ² -Cys ³ -Cys ⁴ -Glu ⁵ -Tyr ⁶ -Cys ^{7 -Cys 8 -Asn 9 -Pro 10 -Ala 11} -Cys ^{12 -Thr 13 -Gly 14} -Cys ¹⁵ - Tyr ¹⁶ - PEG3 82 N24 SZ:S8, S4:S12, S7:S15 PEG3-Asn ¹ -Phe ² -Cys ³ -Cys ⁴ -Glu ⁵ -Tyr ⁶ -Cys ^{7 -Cys 8 -Asn 9 -Pro 10 -Ala 11 -Cys 12 -Thr 13 -Gly 14} -Cys ¹⁵ - Tyr ¹⁶ 83

- 13 040600

N25 SZ:S8, S4:S12, S7:S15 Asn ¹ -Phe ² -Cys ³ -Cys ⁴ -Glu ⁵ -Tyr ^{6 -Cys 7} -Cys 8 -Asn ⁹ -Pro ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Tyr ¹⁶ -PEG3 84 N26 C1:C6, C2:C10, C5:C13 Cys ¹ -Cys ² -Glu3-Ser ⁴ -Cys ⁵ -Cys ⁶ -Asn ⁷ -Pro ⁸ -Ala ⁹ -Cys ¹⁰ -Thr ¹¹ -Gly ¹² -Cys ¹³ -Tyr ¹⁴ 85 N27 C1:C6, C2:C10, C5:C13 Cys ¹ -Cys ² -Glu3-Phe ⁴ -Cys ⁵ -Cys ⁶ -Asn ⁷ -Pro ⁸ -Ala ⁹ -Cys ¹⁰ -Thr ¹¹ -Gly ¹² -Cys ¹³ -Tyr ¹⁴ 86 N28 C1:C6, C2:C10, C5:C13 Cys ¹ -Cys ² -Glu3-Ser ⁴ -Cys ⁵ -Cys ⁶ -Asn ⁷ -Pro ⁸ -Ala ⁹ -Cys ¹⁰ -Thr ¹¹ -Gly ¹² -Cys ¹³ - 87 N29 C1:C6, C2:C10, C5:C13 Cys ¹ -Cys ² -Glu3-Phe ⁴ -Cys ⁵ -Cys ⁶ -Asn ⁷ -Pro ⁸ -Ala ⁹ -Cys ¹⁰ -Thr ¹¹ -Gly ¹² -Cys ¹³ 88 N30 1:6, 2:10,5:13 Pen ¹ -Pen ² -Glu3 -Tyr ⁴ -Pen ⁵ -Pen ⁶ -Asn ⁷ -Pro ⁸ -Ala ⁹ -Pen ¹⁰ -Thr ¹¹ -Gly ¹² -Pen ¹³ -Tyr ¹⁴ 89 N31 1:6, 2:10,5:13 Pen ¹ -Pen ² -Glu3 -Tyr ⁴ -Pen ⁵ -Pen ⁶ -Asn ⁷ -Pro ⁸ -Ala ⁹ -Pen ¹⁰ -Thr ¹¹ -Gly ¹² -Pen ¹³ 90 Formula X C9:C14, C10:C18, S13:S21 Xaa ¹ -Xaa ^2- Xaa ³ -Xaa ⁴ -Xaa ⁵ -Xaa ⁶ -Asn ⁷ -Tyr 8 ^-Cys 9 ^{-Cys 10} -Xaa ¹¹ -Tyr ¹² -Cys ¹³ -Cys ¹⁴ -Xaa ¹⁵ -Xaa ¹⁶ - Xaa ¹⁷ -Cys ¹⁸ -Xaa ¹⁹ -Xaa ²⁰ -Cys ²¹ -Xaa ²² 91 Formula XI S9:S14, S10:S18, S13:S21 Xaa ¹ -Xaa ² ' Xaa ³ -Xaa ⁴ -Xaa ⁵ -Xaa ⁶ -Asn ⁷ -Phe ^{8 -Cys} 9 -Cys ¹⁰ -Xaa ¹¹ -Phe ¹² -Cys ¹³ -Cys ¹⁴ -Xaa ¹⁵ -Xaa ¹⁶ - Xaa ¹⁷ -Cys ¹⁸ -Xaa ¹⁹ -Xaa ²⁰ -Cys ²¹ -Xaa ²² 92 Formula XII SZ:S8, S4:S12, S7:S15 Asn ¹ -Phe ² -Cys ³ -Cys ⁴ -Xaa ⁵ -Phe ⁶ -Cys ⁷ -Cys ⁸ -Xaa ⁹ -Xaa ¹⁰ -Xaa ¹¹ 'Cys ¹² -Xaa ³ -Xaa ¹⁴ 'Cys ¹⁵ -Xaa ¹⁶ 93 Formula XIII 3:8, 4:12, 7:15 Asn ¹ -Phe ² -Pen ³ -Cys ⁴ -Xaa ⁵ -Phe ⁶ -Cys ⁷ Pen ⁸ -Xaa ⁹ -Xaa ¹⁰ -Xaa ¹¹ Cys ¹² -Xaa 13 ^{-Xaa 14 Cys 15} -Xaa ¹⁶ 94 Formula XIV 3:8, 4:12, 7:15 Asn ¹ -Phe ² -Maa ³ -Maa ⁴ -Xaa ⁵ -Xaa ⁶ -Maa ⁷ -Maa ⁸ -Xaa 9 -Xaa ^{10 -Xaa 11} -Maa ¹² -Xaa ¹³ -Xaa ¹⁴ -Maa ¹⁵ -Xaa ¹⁶ 95 Formula XV 1:6, 2:10,5:13 Maa ¹ -Maa ² -Glu3-Xaa ⁴ -Maa ⁵ -Maa ⁶ -Asn ⁷ -Pro ⁸ -Ala ⁹ -Maa ¹⁰ -Thr ¹¹ -Gly ¹² -Maa ¹³ -Tyr ¹⁴ 96 Formula XVI 1:6, 2:10,5:13 Maa ¹ -Maa ² -Glu3-Xaa ⁴ -Maa ⁵ -Maa ⁶ -Asn ⁷ -Pro ⁸ -Ala ⁹ -Maa ¹⁰ -Thr ¹¹ -Gly ¹² -Maa ¹³ 97 Formula XVII 1:6, 2:10,5:13 Xaa _n3 -Maa ¹ -Maa ² -Xaa ³ -Xaa ⁴ -Maa ⁵ -Maa ⁶ -Xaa ⁷ -Xaa ⁸ -Xaa ⁹ -Maa ¹⁰ -Xaa ^{11 -Xaa 12 -Maa 13} -Xaa _n2 98

Table 4

GCRA peptides

Name Position of disulfide bonds Structure SEQIDNO: SP-363 C4:C12,C7:C15 dAsn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val 8 -Asn ^{9 -Val 10} -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dLeu-AMID ¹⁶ dAsn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dSer ¹⁶ 99 SP-364 C4:C12,C7:C15 100 SP-365 C4:C12,C7:C15 dAsn ¹ -Asp ² -Glu ³ -Cys ^{4 -Glu 5} -Leu ⁶ -Cys 7 -Val ⁸ -Asn ⁹ -Val 10 -Ala ^{11 -Cys 12} -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dSer-AMID ¹⁶ 101 SP-366 C4:C12,C7:C15 dAsn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val 8 -Asn ^{9 -Val 10} -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dTyr ¹⁶ dAsn ¹ - Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dTyr-AMID ¹⁶ 102 SP-367 C4:C12,C7:C15 103 SP-373 C4:C12,C7:C15 Pyglu ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn 9 -Val ^{10 -Ala 11} -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dLeu-AMID ¹⁶ 104 / C4:C12,C7:C15 Pyglu ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ^{7 -Val 8 -Asn 9 -Val 10 -Ala 11 -Cys 12 -Thr 13 -Gly 14 -Cys 15} -Leu ¹⁶ PEG3-Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ^{6 -Cys 7} -Val ⁸ -Asn 9 -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Leu ¹⁶ PEG3 251 SP304diPEG C4:C12,C7:C15 105 SP-304NPEG C4:C12,C7:C15 PEG3-Asn ^{1 -Asp 2} -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys 7 -Val 8 -Asn ⁹ -Val ^{10 -Ala 11} -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Leu ¹⁶ 106 SP-304CPEG C4:C12,C7:C15 Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys 7 -Val ^{8 -Asn 9} -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Leu ¹⁶ -PEG3 107

SP-304 analogs, uroguaniline and uroguaniline analogs

Table 5

Name Position of disulfide bonds Structure SEQIDNO Formula XVIII C4:C12, C7:C15 Xaa^Xaa ² - Xaa ³ -Maa ⁴ -Xaa ⁵ -Xaa ⁶ -Maa ⁷ -Xaa ⁸ -Xaa ⁹ -Xaa ¹⁰ -Xaa ⁿ -Maa ^{12 -Xaa 13} -Xaa ¹⁴ -Maa ^{15 -Xaa 16} 108 Uroguaniline C4:C12, C7:C15 Asn ¹ -Asp ² -Asp ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ Cys ¹⁵ -Leu ¹⁶ 109 N32 C4:C12,C7:C15 Glu ¹ -Asp ² -Asp ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Leu ¹⁶ 110 N33 C4:C12,C7:C15 Glu ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Leu ¹⁶ 111 N34 C4:C12, C7:C15 Glu ¹ -Glu ² -Asp ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Leu ¹⁶ 112 N35 C4:C12, C7:C15 Glu ¹ -Glu ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Leu ¹⁶ 113 N36 C4:C12,C7:C15 Asp ¹ -Asp ² -Asp ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Leu ¹⁶ 114 N37 C4:C12,C7:C15 Asp ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Leu ¹⁶ 115 N38 C4:C12,C7:C15 Asp ¹ -Glu ² -Asp ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Leu ¹⁶ 116

- 14 040600

N39 C4:C12,C7:C15 Asp ¹ -Glu ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Leu ¹⁶ 117 N40 C4:C12,C7:C15 Gln ¹ -Asp ² -Asp ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Leu ¹⁶ 118 N41 C4:C12,C7:C15 Gln ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Leu ¹⁶ 119 N42 C4:C12,C7:C15 Gln ¹ -Glu ² -Asp ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Leu ¹⁶ 120 N43 C4:C12,C7:C15 Gln ¹ -Glu ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Leu ¹⁶ 121 N44 C4:C12,C7:C15 Lys ¹ -Asp ² -Asp ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Leu ¹⁶ 122 N45 C4:C12,C7:C15 Lys ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu 6 -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ^{15 -Leu 16} 123 N46 C4:C12,C7:C15 Lys ¹ -Glu ² -Asp ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Leu ¹⁶ 124 N47 C4:C12,C7:C15 Lys ¹ -Glu ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Leu ¹⁶ 125 N48 C4:C12,C7:C15 Glu ¹ -Asp ² -Asp ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Ser ¹⁶ 126 N49 C4:C12,C7:C15 Glu ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Ser ¹⁶ 127 N50 C4:C12,C7:C15 Glu ¹ -Glu ² -Asp ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Ser ¹⁶ 128 N51 C4:C12,C7:C15 Glu ¹ -Glu ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Ser ¹⁶ 129 N52 C4:C12,C7:C15 Asp ¹ -Asp ² -Asp ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Ser ¹⁶ 130 N53 C4:C12,C7:C15 Asp ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Ser ¹⁶ 131 N54 C4:C12,C7:C15 Asp ¹ -Glu ² -Asp ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Ser ¹⁶ 132 N55 C4:C12,C7:C15 Asp ¹ -Glu ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Ser ¹⁶ 133 N56 C4:C12,C7:C15 Gln ¹ -Asp ² -Asp ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Ser ¹⁶ 134 N57 C4:C12,C7:C15 Gln ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val 10 -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ^{15 -Ser 16} 135 N58 C4:C12,C7:C15 Gln ¹ -Glu ² -Asp ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Ser ¹⁶ 136 N59 C4:C12,C7:C15 Gln ¹ -Glu ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Ser ¹⁶ 137 N60 C4:C12,C7:C15 Lys ¹ -Asp ² -Asp ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Ser ¹⁶ 138 N61 C4:C12,C7:C15 Lys ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Ser ¹⁶ 139 N62 C4:C12,C7:C15 Lys ¹ -Glu ² -Asp ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Ser ¹⁶ 140 N63 C4:C12,C7:C15 Lys ¹ -Glu ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Val ⁸ -Asn ⁹ -Val ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Ser ¹⁶ 141 N65 C4:C12,C7:C15 Glu ¹ -Asp ² -Asp ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ^{7 -Ile 8 -Asn 9 -Met 10 -Ala 11} -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Leu ¹⁶ 142 N66 C4:C12,C7:C15 Glu ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ^{7 -Ile 8 -Asn 9} -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Leu ¹⁶ 143 N67 C4:C12,C7:C15 Glu ¹ -Glu ² -Asp ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Leu ¹⁶ 144 N68 C4:C12,C7:C15 Glu ¹ -Glu ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Leu ¹⁶ 145 N69 C4:C12,C7:C15 Asp ¹ -Asp ² -Asp ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Leu ¹⁶ 146 N70 C4:C12,C7:C15 Asp ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Leu ¹⁶ 147 N71 C4:C12,C7:C15 Asp ¹ -Glu ² -Asp ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Leu ¹⁶ 148 N72 C4:C12,C7:C15 Asp ¹ -Glu ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Leu ¹⁶ 149 N73 C4:C12,C7:C15 Gln ¹ -Asp ² -Asp ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Leu ¹⁶ 150 N74 C4:C12,C7:C15 Gln ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Leu ¹⁶ 151 N75 C4:C12,C7:C15 Gln ¹ -Glu ² -Asp ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Leu ¹⁶ 152 N76 C4:C12,C7:C15 Gln ¹ -Glu ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Leu ¹⁶ 153 N77 C4:C12,C7:C15 Lys ¹ -Asp ² -Asp ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ^{7 -Ile 8 -Asn 9} -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Leu ¹⁶ 154 N78 C4:C12,C7:C15 Lys ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ^{7 -Ile 8 -Asn 9} -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Leu ¹⁶ 155 N79 C4:C12,C7:C15 Lys ¹ -Glu ² -Asp ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Leu ¹⁶ 156 N80 C4:C12,C7:C15 Lys ¹ -Glu ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Leu ¹⁶ 157 N81 C4:C12,C7:C15 Glu ¹ -Asp ² -Asp ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Ser ¹⁶ 158 N82 C4:C12,C7:C15 Glu ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Ser ¹⁶ 159 N83 C4:C12,C7:C15 Glu ¹ -Glu ² -Asp ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Ser ¹⁶ 160 N84 C4:C12,C7:C15 Glu ¹ -Glu ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Ser ¹⁶ 161 N85 C4:C12,C7:C15 Asp ¹ -Asp ² -Asp ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Ser ¹⁶ 162 N86 C4:C12,C7:C15 Asp ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Ser ¹⁶ 163 N87 C4:C12,C7:C15 Asp ¹ -Glu ² -Asp ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Ser ¹⁶ 164 N88 C4:C12,C7:C15 Asp ¹ -Glu ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Ser ¹⁶ 165 N89 C4:C12,C7:C15 Gln ¹ -Asp ² -Asp ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ^{10 -Ala 11} -Cys ¹² -Thr ¹³ -Gly ^{14 -Cys 15} -Ser ¹⁶ 166 N90 C4:C12,C7:C15 Gln ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met 10 -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ^{14 -Cys 15} -Ser ¹⁶ 167 N91 C4:C12,C7:C15 Gln ¹ -Glu ² -Asp ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met 10 -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ^{15 -Ser 16} 168 N92 C4:C12,C7:C15 Gln ¹ -Glu ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ^{10 -Ala 11} -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ^{15 -Ser 16} 169 N93 C4:C12,C7:C15 Lys ¹ -Asp ² -Asp ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Ser ¹⁶ 170 N94 C4:C12,C7:C15 Lys ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Ser ¹⁶ 171 N95 C4:C12,C7:C15 Lys ¹ -Glu ² -Asp ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Ser ¹⁶ 172 N96 C4:C12,C7:C15 Lys ¹ -Glu ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Ser ¹⁶ 173

- 15 040600

Table 6

Guaniline and analogues

Name Position disulfide bonds Structure SEQIDNO Formula XIX 4:12,7:15 Xaa^Xaa ² - Xaa ³ -Maa ⁴ -Xaa ⁵ -Xaa ⁶ -Maa ⁷ -Xaa ⁸ -Xaa ⁹ -Xaa ¹⁰ -Xaa ¹¹ -Maa ¹² -Xaa ¹³ -Xaa ¹⁴ -Maa ¹⁵ 174 Guanylin C4:C12, C7:C15 Ser ¹ -His ² -Thr ³ -Cys ⁴ -Glu ⁵ -Ile ⁶ -Cys ⁷ -Ala ⁸ -Phe ⁹ -Ala ¹⁰ -Ala ¹¹ -Cys ¹² -Ala ¹³ -Gly ¹⁴ -Cys ¹⁵ 175 Human Guanylin C4:C12, C7:C15 Pro ¹ -Gly ² -Thr ³ -Cys ⁴ -Glu ⁵ -Ile ⁶ -Cys ⁷ -Ala ⁸ -Tyr ⁹ -Ala ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ 252 N97 C4:C12, C7:C15 Ser ¹ -His ² -Thr ³ -Cys ⁴ -Glu ⁵ -Ile ⁶ -Cys ⁷ -Ala ⁸ -Asn ⁹ -Ala ¹⁰ -Ala ¹¹ -Cys ¹² -Ala ¹³ -Gly ¹⁴ -Cys ¹⁵ 176 N98 C4:C12, C7:C15 Ser ¹ -His ² -Thr ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Ala ⁸ -Asn ⁹ -Ala ¹⁰ -Ala ¹¹ -Cys ¹² -Ala ¹³ -Gly ¹⁴ -Cys ¹⁵ 177 N99 C4:C12, C7:C15 Ser ¹ -His ² -Thr ³ -Cys ⁴ -Glu ⁵ -Val ⁶ -Cys ⁷ -Ala ⁸ -Asn ⁹ -Ala ¹⁰ -Ala ¹¹ -Cys ¹² -Ala ¹³ -Gly ¹⁴ -Cys ¹⁵ 178 N100 C4:C12,C7:C15 Ser ¹ -His ² -Thr ³ -Cys ⁴ -Glu ⁵ -Tyr ⁶ -Cys ⁷ -Ala ⁸ -Asn ⁹ -Ala ¹⁰ -Ala ¹¹ -Cys ¹² -Ala ¹³ -Gly ¹⁴ -Cys ¹⁵ 179 N101 C4:C12,C7:C15 Ser ¹ -His ² -Thr ³ -Cys ⁴ -Glu ⁵ -Ile ⁶ -Cys ⁷ -Ala ⁸ -Asn ⁹ -Ala ¹⁰ -Ala ¹¹ -Cys ¹² -Ala ¹³ -Gly ¹⁴ -Cys ¹⁵ 180 N102 C4:C12, C7:C15 Ser ¹ -His ² -Thr ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Ala ⁸ -Asn ⁹ -Ala ¹⁰ -Ala ¹¹ -Cys ¹² -Ala ¹³ -Gly ¹⁴ -Cys ¹⁵ 181 N103 C4:C12,C7:C15 Ser ¹ -His ² -Thr ³ -Cys ⁴ -Glu ⁵ -Val ⁶ -Cys ⁷ -Ala ⁸ -Asn ⁹ -Ala ¹⁰ -Ala ¹¹ -Cys ¹² -Ala ¹³ -Gly ¹⁴ -Cys ¹⁵ 182 N104 C4:C12, C7:C15 Ser ¹ -His ² -Thr ³ -Cys ⁴ -Glu ⁵ -Tyr ⁶ -Cys ⁷ -Ala ⁸ -Asn ⁹ -Ala ¹⁰ -Ala ¹¹ -Cys ¹² -Ala ¹³ -Gly ¹⁴ -Cys ¹⁵ 183 N105 C4:C12, C7:C15 Ser ¹ -His ² -Thr ³ -Cys ⁴ -Glu ⁵ -Ile ⁶ -Cys ⁷ -Ala ⁸ -Asn ⁹ -Ala ¹⁰ -Ala ¹¹ -Cys ¹² -Ala ¹³ -Gly ¹⁴ -Cys ¹⁵ 184 N106 C4:C12, C7:C15 Ser ¹ -His ² -Thr ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Ala ⁸ -Asn ⁹ -Ala ¹⁰ -Ala ¹¹ -Cys ¹² -Ala ¹³ -Gly ¹⁴ -Cys ¹⁵ 185 N107 C4:C12,C7:C15 Ser ¹ -His ² -Thr ³ -Cys ⁴ -Glu ⁵ -Val ⁶ -Cys ⁷ -Ala ⁸ -Asn ⁹ -Ala ¹⁰ -Ala ¹¹ -Cys ¹² -Ala ¹³ -Gly ¹⁴ -Cys ¹⁵ 186 N108 C4:C12,C7:C15 Ser ¹ -His ² -Thr ³ -Cys ⁴ -Glu ⁵ -Tyr ⁶ -Cys ⁷ -Ala ⁸ -Asn ⁹ -Ala ¹⁰ -Ala ¹¹ -Cys ¹² -Ala ¹³ -Gly ¹⁴ -Cys ¹⁵ 187 N109 C4:C12,C7:C15 Ser ¹ -His ² -Thr ³ -Cys ⁴ -Glu ⁵ -Ile ⁶ -Cys ⁷ -Ala ⁸ -Asn ⁹ -Ala ¹⁰ -Ala ¹¹ -Cys ¹² -Ala ¹³ -Gly ¹⁴ -Cys ¹⁵ 188 N110 C4:C12, C7:C15 Ser ¹ -His ² -Thr ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Ala ⁸ -Asn ⁹ -Ala ¹⁰ -Ala ¹¹ -Cys ¹² -Ala ¹³ -Gly ¹⁴ -Cys ¹⁵ 189 N111 C4:C12, C7:C15 Ser ¹ -His ² -Thr ³ -Cys ⁴ -Glu ⁵ -Val ⁶ -Cys ⁷ -Ala ⁸ -Asn ⁹ -Ala ¹⁰ -Ala ¹¹ -Cys ¹² -Ala ¹³ -Gly ¹⁴ -Cys ¹⁵ 190 N112 C4:C12,C7:C15 Ser ¹ -His ² -Thr ³ -Cys ⁴ -Glu ⁵ -Tyr ⁶ -Cys ⁷ -Ala ⁸ -Asn ⁹ -Ala ¹⁰ -Ala ¹¹ -Cys ¹² -Ala ¹³ -Gly ¹⁴ -Cys ¹⁵ 191 N113 C4:C12, C7:C15 Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Ile ⁶ -Cys ⁷ -Ala ⁸ -Asn ⁹ -Ala ¹⁰ -Ala ¹¹ -Cys ¹² -Ala ¹³ -Gly ¹⁴ -Cys ¹⁵ 192 N114 C4:C12,C7:C15 Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Ala ⁸ -Asn ⁹ -Ala ¹⁰ -Ala ¹¹ -Cys ¹² -Ala ¹³ -Gly ¹⁴ -Cys ¹⁵ 193 N115 C4:C12, C7:C15 Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Val ⁶ -Cys ⁷ -Ala ⁸ -Asn ⁹ -Ala ¹⁰ -Ala ¹¹ -Cys ¹² -Ala ¹³ -Gly ¹⁴ -Cys ¹⁵ 194 N116 C4:C12,C7:C15 Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Tyr ⁶ -Cys ⁷ -Ala ⁸ -Asn ⁹ -Ala ¹⁰ -Ala ¹¹ -Cys ¹² -Ala ¹³ -Gly ¹⁴ -Cys ¹⁵ 195 N117 C4:C12,C7:C15 Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Ile ⁶ -Cys ⁷ -Ala ⁸ -Asn ⁹ -Ala ¹⁰ -Ala ¹¹ -Cys ¹² -Ala ¹³ -Gly ¹⁴ -Cys ¹⁵ 196 N118 C4:C12, C7:C15 Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Ala ⁸ -Asn ⁹ -Ala ¹⁰ -Ala ¹¹ -Cys ¹² -Ala ¹³ -Gly ¹⁴ -Cys ¹⁵ 197 N119 C4:C12, C7:C15 Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Val ⁶ -Cys ⁷ -Ala ⁸ -Asn ⁹ -Ala ¹⁰ -Ala ¹¹ -Cys ¹² -Ala ¹³ -Gly ¹⁴ -Cys ¹⁵ 198 N120 C4:C12,C7:C15 Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Tyr ⁶ -Cys ⁷ -Ala ⁸ -Asn ⁹ -Ala ¹⁰ -Ala ¹¹ -Cys ¹² -Ala ¹³ -Gly ¹⁴ -Cys ¹⁵ 199 N121 C4:C12,C7:C15 Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Ile ⁶ -Cys ⁷ -Ala ⁸ -Asn ⁹ -Ala ¹⁰ -Ala ¹¹ -Cys ¹² -Ala ¹³ -Gly ¹⁴ -Cys ¹⁵ 200 N122 C4:C12, C7:C15 Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Ala ⁸ -Asn ⁹ -Ala ¹⁰ -Ala ¹¹ -Cys ¹² -Ala ¹³ -Gly ¹⁴ -Cys ¹⁵ 201 N123 C4:C12, C7:C15 Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Val ⁶ -Cys ⁷ -Ala ⁸ -Asn ⁹ -Ala ¹⁰ -Ala ¹¹ -Cys ¹² -Ala ¹³ -Gly ¹⁴ -Cys ¹⁵ 202 N124 C4:C12, C7:C15 Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Tyr ⁶ -Cys ⁷ -Ala ⁸ -Asn ⁹ -Ala ¹⁰ -Ala ¹¹ -Cys ¹² -Ala ¹³ -Gly ¹⁴ -Cys ¹⁵ 203 N125 C4:C12, C7:C15 Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Ile ⁶ -Cys ⁷ -Ala ⁸ -Asn ⁹ -Ala ¹⁰ -Ala ¹¹ -Cys ¹² -Ala ¹³ -Gly ¹⁴ -Cys ¹⁵ 204 N126 C4:C12, C7:C15 Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Ala ⁸ -Asn ⁹ -Ala ¹⁰ -Ala ¹¹ -Cys ¹² -Ala ¹³ -Gly ¹⁴ -Cys ¹⁵ 205 N127 C4:C12,C7:C15 Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Val ⁶ -Cys ⁷ -Ala ⁸ -Asn ⁹ -Ala ¹⁰ -Ala ¹¹ -Cys ¹² -Ala ¹³ -Gly ¹⁴ -Cys ¹⁵ 206 N128 C4:C12, C7:C15 Asn ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Tyr ⁶ -Cys ⁷ -Ala ⁸ -Asn ⁹ -Ala ¹⁰ -Ala ¹¹ -Cys ¹² -Ala ¹³ -Gly ¹⁴ -Cys ¹⁵ 207

- 16 040600

Lymphoguaniline and analogues

Table 7

Name Position of disulfide bonds Structure SEQIDNO Formula XX 4:12 Xaa^Xaa ² - Xaa ³ -Maa ⁴ -Xaa ⁵ -Xaa ⁶ -Maa ⁷ -Xaa ⁸ -Xaa ⁹ -Xaa ¹⁰ -Xaa ¹¹ -Maa ^{12 -Xaa 13 -Xaa 14 -Xaa} _n1 ¹⁵ 208 Lymphoguaniline C4:C12 Gln ¹ -Glu ² -Glu- ³ Cys ⁴ -Glu ⁵ -Leu ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Tyr ¹⁵ 209 N129 C4:C12 Gln ¹ -Glu ² -Glu ³ -Cys ⁴ -Glu ⁵ -Thr ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Tyr ¹⁵ 210 N130 C4:C12 Gln ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Thr ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Tyr ¹⁵ 211 N131 C4:C12 Gln ¹ -Asp ² -Asp ³ -Cys ⁴ -Glu ⁵ -Thr ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Tyr ¹⁵ 212 N132 C4:C12 Gln ¹ -Glu ² -Asp ³ -Cys ⁴ -Glu ⁵ -Thr ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Tyr ¹⁵ 213 N133 C4:C12 Gln ¹ -Glu ² -Glu ³ -Cys ⁴ -Glu ⁵ -Glu ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Tyr ¹⁵ 214 N134 C4:C12 Gln ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Glu ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Tyr ¹⁵ 215 N135 C4:C12 Gln ¹ -Asp ² -Asp ³ -Cys ⁴ -Glu ⁵ -Glu ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Tyr ¹⁵ 216 N136 C4:C12 Gln ¹ -Glu ² -Asp ³ -Cys ⁴ -Glu ⁵ -Glu ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Tyr ¹⁵ 217 N137 C4:C12 Gln ¹ -Glu ² -Glu ³ -Cys ⁴ -Glu ⁵ -Tyr ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Tyr ¹⁵ 218 N138 C4:C12 Gln ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Tyr ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Tyr ¹⁵ 219 N139 C4:C12 Gln ¹ -Asp ² -Asp ³ -Cys ⁴ -Glu ⁵ -Tyr ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Tyr ¹⁵ 220 N140 C4:C12 Gln ¹ -Glu ² -Asp ³ -Cys ⁴ -Glu ⁵ -Tyr ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Tyr ¹⁵ 221 N141 C4:C12 Gln ¹ -Glu ² -Glu ³ -Cys ⁴ -Glu ⁵ -Ile ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Tyr ¹⁵ 222 N142 C4:C12 Gln ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Ile ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Tyr ¹⁵ 223 N143 C4:C12 Gln ¹ -Asp ² -Asp ³ -Cys ⁴ -Glu ⁵ -Ile ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Tyr ¹⁵ 224 N144 C4:C12 Gln ¹ -Glu ² -Asp ³ -Cys ⁴ -Glu ⁵ -Ile ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Tyr ¹⁵ 225 N145 C4:C12,C7:C15 Gln ¹ -Glu ² -Glu ³ -Cys ⁴ -Glu ⁵ -Thr ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met 10 -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ^{15 -Ser 16} 226 N146 C4:C12,C7:C15 Gln ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Thr ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met 10 -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ^{15 -Ser 16} 227 N147 C4:C12, C7:C15 Gln ¹ -Asp ² -Asp ³ -Cys ⁴ -Glu ⁵ -Thr ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met 10 -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ^{15 -Ser 16} 228 N148 C4:C12, C7:C15 Gln ¹ -Glu ² -Asp ³ -Cys ⁴ -Glu ⁵ -Thr ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ^{10 -Ala 11} -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ^{15 -Ser 16} 229 N149 C4:C12,C7:C15 Gln ¹ -Glu ² -Glu ³ -Cys ⁴ -Glu ⁵ -Glu ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met 10 -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ^{15 -Ser 16} 230 N150 C4:C12, C7:C15 Gln ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Glu ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Ser 231 N151 C4:C12, C7:C15 Gln ¹ -Asp ² -Asp ³ -Cys ⁴ -Glu ⁵ -Glu ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met 10 -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ^{14 -Cys 15} -Ser ¹⁶ 232 N152 C4:C12, C7:C15 Gln ¹ -Glu ² -Asp ³ -Cys ⁴ -Glu ⁵ -Glu ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met 10 -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ^{15 -Ser 16} 233 N153 C4:C12,C7:C15 Gln ¹ -Glu ² -Glu ³ -Cys ⁴ -Glu ⁵ -Tyr ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met 10 -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ^{15 -Ser 16} 234 N154 C4:C12, C7:C15 Gln ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Tyr ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met 10 -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ^{15 -Ser 16} 235 N155 C4:C12,C7:C15 Gln ¹ -Asp ² -Asp ³ -Cys ⁴ -Glu ⁵ -Tyr ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met 10 -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ^{15 -Ser 16} 236 N156 C4:C12, C7:C15 Gln ¹ -Glu ² -Asp ³ -Cys ⁴ -Glu ⁵ -Tyr ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met 10 -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ^{15 -Ser 16} 237 N157 C4:C12,C7:C15 Gln ¹ -Glu ² -Glu ³ -Cys ⁴ -Glu ⁵ -Ile ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met ¹⁰ -Ala 11 -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ^{15 -Ser 16} 238 N158 C4:C12, C7:C15 Gln ¹ -Asp ² -Glu ³ -Cys ⁴ -Glu ⁵ -Ile ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met 10 -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ^{15 -Ser 16} 239 N159 C4:C12,C7:C15 Gln ¹ -Asp ² -Asp ³ -Cys ⁴ -Glu ⁵ -Ile ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met 10 -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ^{15 -Ser 16} 240 N160 C4:C12,C7:C15 Gln ¹ -Glu ² -Asp ³ -Cys ⁴ -Glu ⁵ -Ile ⁶ -Cys ⁷ -Ile ⁸ -Asn ⁹ -Met 10 -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ^{15 -Ser 16} 241

ST Peptide and analogues

Table 8

Name Position of disulfide bonds Structure SEQIDNO ST Peptide C9:C14,C10:C18,C13:C21 Asn ¹ -Ser ² -Ser ³ -Asn ⁴ -Ser ⁵ -Ser ⁶ -Asn ^{7 -Tyr 8} -Cys 9 -Cys ¹⁰ -Glu ¹¹ -Lys ¹² -Cys ¹³ -Cys ¹⁴ -Asn ¹⁵ -Pro ¹⁶ -Ala ¹⁷ - 242 Cys ¹⁸ -Thr ¹⁹ -Gly ²⁰ -Cys ²¹ -Tyr ²² N161 SZ:S8,S4:S12,S7:S15 PEG3-Asn ¹ -Phe ² -Cys ³ -Cys ⁴ -Glu ⁵ -Thr ⁶ -Cys ⁷ -Cys 8 -Asn ⁹ -Pro ^{10 -Ala 11} -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Tyr ¹⁶ - PEG3 243 N162 SZ:S8,S4:S12,S7:S15 PEG3- ^{Asn 1} -Phe ² -Cys ³ -Cys ⁴ -Glu ⁵ -Thr ⁶ -Cys ⁷ -Cys 8 -Asn 9 -Pro ^{10 -Ala 11} -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Tyr ¹⁶ 244 N163 SZ:S8,S4:S12,S7:S15 Asn ¹ -Phe ² -Cys ³ -Cys ⁴ -Glu ⁵ -Thr ⁶ -Cys ⁷ -Cys ⁸ -Asn ⁹ -Pro ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Tyr ¹⁶ -PEG3 245 N164 SZ:S8,S4:S12,S7:S15 Asn ¹ -Phe ² -Cys ³ -Cys ⁴ -Glu ⁵ -Tyr ⁶ -Cys ⁷ -Cys ⁸ -Asn ⁹ -Pro ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Tyr ¹⁶ 246 N165 SZ:S8,S4:S12,S7:S15 dAsn ¹ -Phe ² -Cys ³ -Cys ⁴ -Glu ⁵ -Tyr ^{6 -Cys} 7 -Cys ⁸ -Asn ⁹ -Pro ^{10 -Ala 11} -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dTyr ¹⁶ 247 N166 SZ:S8,S4:S12,S7:S15 Asn ¹ -Phe ² -Cys ³ -Cys ⁴ -Glu ⁵ -Tyr ⁶ -Cys ⁷ -Cys ⁸ -Asn ⁹ -Pro ¹⁰ -Ala ¹¹ -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -dTyr ¹⁶ 248 N167 SZ:S8,S4:S12,S7:S15 dAsn ¹ -Phe ² -Cys ³ -Cys ⁴ -Glu ⁵ -Tyr ^{6 -Cys 7} -Cys 8 -Asn ⁹ -Pro ^{10 -Ala 11} -Cys ¹² -Thr ¹³ -Gly ¹⁴ -Cys ¹⁵ -Tyr ¹⁶ 249

The term essentially consisting of includes peptides that are identical to the recited sequence (any of Tables 2-8) and other sequences that do not substantially differ in terms of either structure or function. For the purposes of the present application, a peptide differs substantially if its structure differs by more than three amino acids from any of the peptides in Table. 2-8 or if its activation of cellular cGMP production is reduced or increased by more than

50%. Preferably, substantially similar peptides should differ by no more than two amino acids and not differ by more than about 25% in terms of activation of cGMP production.

Also included within the meaning of substantially homologous is any GCRA peptide that can be isolated by cross-reactivity with antibodies in the GCRA peptide.

Obtaining peptides-GCRA.

The GCRA peptides are readily prepared using modern cloning techniques or can be synthesized using solid phase methods or site-directed mutagenesis. The GCRA peptide may include dominant negative forms of the polypeptide.

Chemical synthesis can generally be carried out using standard liquid phase or solid phase peptide synthesis techniques in which the peptide bond is formed through direct condensation of the amino group of one amino acid with the carboxy group of another amino acid, eliminating the water molecule. The synthesis of a peptide bond by direct condensation, as formulated above, requires suppression of the reactive nature of the amino group of the first and carboxyl groups of the second amino acids. The masking substituents should ensure their easy removal without causing disruption of the unstable peptide molecule.

A wide range of coupling methods and protecting groups can be used in liquid phase synthesis (See, Gross and Meienhofer, eds., The Peptides: Analysis, Synthesis, Biology, vol. 1-4 (Academic Press, 1979); Bodansky and Bodansky, The Practice of Peptide Synthesis, 2d ed (Springer Verlag, 1994)).

In addition, intermediate cleaning and linear upscaling are possible. Those of ordinary skill in the art will appreciate that solution synthesis requires consideration of the backbone and side chain protecting groups and the activation method. In addition, careful selection of segments is necessary to minimize racemization during segment condensation. Solubility considerations are also a factor. Solid phase peptide synthesis uses an insoluble polymer for support during organic synthesis. The polymer-supported peptide chain allows simple washing and filtration steps to be used instead of time-consuming cleanups in intermediate steps. Solid phase peptide synthesis can generally be carried out according to the method of Merrifield et al., J. Am. Chem. Soc, 1963, 85:2149, which involves the assembly of a linear peptide chain on a polymeric support using protected amino acids. Solid phase peptide synthesis typically uses either the Boc or Fmoc strategies, which are well known in the art.

Those of ordinary skill in the art will appreciate that in solid phase synthesis, the deprotection and coupling reactions must proceed to completion and the side chain blocking groups must be stable throughout the synthesis. In addition, solid phase synthesis is usually the most suitable when peptides need to be produced on a small scale.

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

Alternatively, GCRA peptides are obtained using modern cloning techniques. For example, GCRA peptides are produced either in bacteria, including but not limited to E. coli, or in other existing systems for producing polypeptides or proteins (e.g., Bacillus subtilis, baculovirus expression systems using Drosophila Sf9 cells, yeast or filamentous fungus expression systems). , mammalian cellular expression systems), or they can be chemically synthesized. If the GCRA peptide or other peptide is to be generated in bacteria, eg E. coli, the nucleic acid molecule encoding the polypeptide may also encode a leader sequence that allows the mature polypeptide to be secreted from the cell. Thus, a polypeptide coding sequence may comprise a presequence and a prosequence of, for example, a naturally occurring bacterial ST polypeptide. The secreted, mature polypeptide can be purified from the culture medium.

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

Many transcription control sequences are well known to those skilled in the art. The expression vector may also contain a translational regulatory sequence (eg, a 5' untranslated sequence, a 3' untranslated sequence, or an internal ribosome entry site).

The vector may be capable of autonomous replication, or it may integrate into the host's DNA to provide stability during polypeptide production.

A protein coding sequence that contains the GCRA peptide described herein can also be fused to a nucleic acid encoding a polypeptide affinity marker, e.g., glutathione S-transferase (GST), maltose E binding protein, protein A, FLAG marker, hexa -histidine, mycological marker or HA marker of influenza, in order to facilitate cleaning. An affinity marker or reporter fusion links the reading frame of the polypeptide of interest to the reading frame of the gene encoding the affinity marker, such that a translational fusion is generated. Expression of the fusion gene results in translation of a single polypeptide that contains both the polypeptide of interest and the affinity marker. In some cases where affinity markers are used, the DNA sequence encoding the protease recognition site will fuse between the reading frames for the affinity marker and the polypeptide of interest.

Genetic constructs and methods suitable for generating immature and mature forms of the peptides and GCRA variants described herein in non-bacterial protein expression systems, and well known to those skilled in the art, can also be used to generate polypeptides in a biological system. .

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

By inhibition or suppression or reduction is meant that the GCRA peptide reduces protein activity and/or production by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 100% or more relative to activity and/or protein production without the GCRA peptide.

By inducing is meant that the GCRA peptide increases activity and/or protein production by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least

40%, at least 45%, at least 50%, at least 55%, at least

60%, at least 65%, at least 70%, at least 75%, at least

80%, at least 85%, at least 90%, at least 95%, or at least 100% or more relative to activity and/or protein production without the GCRA peptide.

The term treatment or treatment refers to reducing or alleviating symptoms in a patient, preventing worsening or worsening of symptoms, and/or preventing a disease in a patient who does not have it. For a given patient, improvement, worsening, amelioration, or worsening of symptoms can be determined by any objective or subjective criterion. The effectiveness of a treatment can be measured as an improvement in morbidity or mortality (eg, a lengthening of the survival curve for a selected population). Thus, effective treatment should include treating the existing disease, controlling the disease by slowing or stopping its progression, preventing the onset of the disease, reducing the number or severity of symptoms, or a combination thereof. The result can be shown at the control stage using one or more statistically significant criteria.

The term “prevention” in relation to a given disease or disorder means preventing the onset of the development of the disease if the onset of the disease or disorder has not been prevented in a patient who may be predisposed to the disorder or disease but who has not yet been diagnosed as having the disorder or disease, and/or preventing further development of the disease/disorder, if it already exists.

Intracellular cGMP is produced by exposure to, for example, tissue (eg, gastrointestinal tissue, lung tissue, esophageal tissue) or cell contact with GCRA agonists. By induction is meant an increase in the production of cGMP compared to a tissue or cell that has not been in contact with a peptide or GCRA variant. The tissues or cells are directly contacted with the peptide or GCRA variant. Alternatively, the GCRA peptide or variant is administered systemically. The peptide or GCRA variant is administered in an amount sufficient to increase the intracellular concentration of cGMP. The production of cGMP is measured by cell assay known in the art (25).

Disorders are treated, prevented or alleviated by the administration to the person in need of it.

- 19 040600 to a patient, for example a mammal such as a human, a therapeutically effective dose of the GCRA peptide. The GCRA peptides may be present in the pharmaceutical composition as a single dose, along with one or more pharmaceutically acceptable excipients. The term unit dose form refers to a single drug delivery object, such as a tablet, capsule, solution, or inhalation dosage form. The amount of peptide present should be sufficient to have a positive therapeutic effect when administered to a patient (typically between 10 μg and 3 g). What constitutes a positive therapeutic effect will depend on the particular pathological condition being treated and will include any significant improvement in the pathological condition readily recognizable by one of skill in the art.

Peptides-GCRA can be administered alone or in combination with an opioid. Opioids used for pain relief are known in the art. For example, opioid compounds include, but are not limited to, alfentanil, anileridine, azimadolin, bremazocine, burprenorphine, butorphanol, codeine, dezocin, diacetylmorphine (heroin), dihydrocodeine, diphenoxylate, ethylmorphine, fedotosine, fentanyl, funaltrexamine, hydrocodone, hydromorphone, levallorphan, levomethadil acetate, levorphanol, loperamide, meperidine (pethidine), methadone, morphine, morphine-6-glucuronide, nalbuphine, nalorphine, nymorphine, opium, oxycodone, oxymorphone, pantopon, pentazocine, propyram, propoxyphene, remifentanil, sufentanil, tilidine, trimebutine, and tramadol . In some embodiments, the opioid is at least one opioid selected from alfentanil, buprenorphine, butorphanol, codeine, deocine, dihydrocodeine, fentanyl, hydrocodone, hydromorphone, levorphanol, meperidine (pethidine), methadone, morphine, nalbuphine, nicomorphine, oxycodone, oxymorphone , pantopon, pentazocine, propiram, propoxyphene, sufentanil and/or tramadol. In some embodiments of the present invention, the opioid is selected from morphine, codeine, oxycodone, hydrocodone, dihydrocodeine, propoxyphene, fentanyl, tramadol, and mixtures thereof. In a particular embodiment, the opioid is loperamide. In other embodiments, the opioid is a mixed agonist such as butorphanol. In some embodiments, patients are administered more than one opioid, such as morphine and heroin or methadone and heroin.

The GCRA peptides can be administered alone or in combination with another therapeutic agent, such as therapeutic agents for the treatment, prevention, or amelioration of symptoms of opioid-induced dysfunction. Illustrative therapeutic agents include, but are not limited to, oral laxatives (eg, emollients or motility-inducing agents), suppositories, enemas, opioid antagonists (eg, naloxone, nalmefene, methylnaltrexone), polyethylene glycol, drugs that selectively release peripheral mu-opioid receptors ( eg, methylnaltrexone, alvimopan), type 2 chloride ion channel activators (eg, lubiprostone), dopamine receptor antagonists (eg, metoclopramide), neoopioids, 5-hydroxytryptamine receptor ligands (serotonin, 5HT), ampakines, K receptor inhibitors -methyl-O-aspartate, AMPA receptor modulators, tapendatol, NKTR-118, TD-1211 and/or microglial inhibitors (eg minocycline).

The term combination therapy means the administration of two or more active agents simultaneously or sequentially. Simultaneous administration can be achieved with a dosage form in which two or more active agents are mixed or with the simultaneous administration of two or more active agents prepared in independent dosage forms. Sequential administration of two or more active agents can be achieved with two or more active agents formulated in independent dosage forms administered sequentially, with one agent administered first followed by a second agent administered seconds, minutes, hours or days after the first agent. .

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

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

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

Combination therapy may also include the administration of two or more agents through different routes or sites. For example, (a) one agent is administered orally and the other agents are administered intravenously, or (b) one agent is administered orally and the other is administered topically. In each case, the agents may be administered either simultaneously or sequentially. Approximate dosages for some of the combination therapy agents described herein can be found in the BNF Recommended Dose column of the tables on pages 11-17 of WO 01/76632 (data in the tables refer to March 2000 British National Formulary) and can also be found in other standard formularies and other prescribing catalogues. For some drugs, the usual prescribed dose for the indication will vary slightly from country to country.

The GCRA peptides, alone or in combination, may be combined with any pharmaceutically acceptable carrier or vehicle. Thus, they can be combined with materials that do not create adverse, allergic or other undesirable reactions when administered to a patient. Carriers or media used may include solvents, dispersants, absorption coatings, controlled release agents, and one or more inert excipients (which include starches, polyols, granulating agents, microcrystalline cellulose (e.g., Celphere beads®), diluents , lubricants, binders, tablet disintegrating agents, etc.), etc. If desired, tablet dosages of the disclosed compositions may be coated using standard aqueous or non-aqueous techniques.

The pharmaceutical composition of the invention is formulated into a dosage form compatible with its intended route of administration. Examples of routes of administration include parenteral, eg, intravenous, intradermal, subcutaneous, oral (eg, inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous administration may include the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and tonicity adjusting agents such as sodium chloride or dextrose. The pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. The parenteral preparation may be enclosed in ampoules, disposable syringes or multi-dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (when soluble in water) or dispersions and sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable vehicles include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS). In all cases, the composition must be sterile and must be fluid to the extent that easy syringability is possible. It must be stable under the conditions of manufacture and storage and must be protected against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg, glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Suitable fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using surfactants. Prevention of the action of microorganisms can be achieved with various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases it will be preferable to include isotonic agents, eg sugars, polyalcohols such as mannitol, sorbitol, sodium chloride, in the composition. Prolonged absorption of injectable compositions can be caused by the inclusion in the composition of an agent that delays absorption, such as aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound (eg, a GCRA agonist) in the required amount in a suitable solvent with one or a combination of the ingredients listed above, as required, followed by filtered sterilization. In general, dispersions are prepared by incorporating the active compound into a sterile medium which contains the basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for preparing sterile injectable solutions, the preparation methods are vacuum drying and freeze drying, which yield a powder of the active ingredient plus any additional desired ingredient from its pre-sterilized and filtered solution.

Oral compositions typically include an inert diluent or food carrier such as mannitol, fructooligosaccharides, polyethylene glycol, and other excipients. They may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be combined with excipients and used in the form of tablets, lozenges or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthrinse, wherein the compound in the fluid carrier is administered orally, rinsed, and spit out or swallowed. Pharmaceutically compatible binding agents and/or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a tablet disintegrating agent such as alginic acid, Primogel or cornstarch; a lubricant such as magnesium stearate or sterotes; a glidant such as colloidal silicon dioxide; a sweetener such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavor.

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressurized container or dispenser that contains a suitable propellant, for example a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, dosage forms use penetrating agents suitable for the barrier to be passed through. Such penetrants are generally known in the art and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be carried out through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated as ointments, salves, gels or creams, as is generally known in the art.

The compounds may also be prepared in the form of suppositories (eg, with conventional suppository bases such as coconut oil and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are formulated with carriers that will protect the compound from rapid elimination from the body, such as controlled release dosage forms, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for obtaining such dosage forms will be clear to those skilled in the art. Materials may also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. They can be prepared according to methods known to those skilled in the art, for example, as described in US Pat. No. 4,522,811, incorporated herein by reference in its entirety.

For ease of administration and dosage uniformity, it is particularly preferred to formulate oral or parenteral compositions in unit dosage form. Dosage unit form, as used herein, refers to physically discrete units suitable as unit dosages for the patient to be treated, each unit containing a predetermined amount of the active compound calculated to produce the desired therapeutic effect in combination with the desired pharmaceutical carrier. The description of unit dosage forms of the invention is dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved.

Pharmaceutical compositions may be enclosed in a container, pack or dispenser along with instructions for administration.

The compositions of the present invention may also optionally include other therapeutic ingredients, anti-caking agents, preservatives, sweetening agents, coloring agents, flavoring agents, drying agents, plasticizers, coloring agents, glidants, anti-adherents, antistatic agents, surfactants (wetting agents) , antioxidants, film-coating agents, and the like. Any such optional ingredient must be compatible with the compound described herein in order to

- 22 040600 ensuring the stability of the dosage form.

The composition may contain other excipients as needed, including, for example, lactose, glucose, fructose, galactose, trehalose, sucrose, maltose, raffinose, maltitol, melicitose, stachyose, lactitol, palatinit, starch, xylitol, mannitol, myoinositol, etc. P. and their hydrates, and amino acids, such as alanine, glycine and betaine, and polypeptides and proteins, such as albumin.

Examples of excipients for use as pharmaceutically acceptable carriers and pharmaceutically acceptable inert carriers and the aforementioned additional ingredients include, but are not limited to, binders, fillers, disintegrants, lubricants, antimicrobial agents, and coating agents such as the following.

Binders: corn starch, potato starch, other starches, gelatin, natural and synthetic gums such as acacia, xanthan gum, sodium alginate, alginic acid, other alginates, ground gum tragacanth, guar gum, cellulose and its derivatives (e.g. ethyl cellulose, cellulose acetate, calcium carboxymethylcellulose, sodium carboxymethylcellulose), polyvinylpyrrolidone (e.g. povidone, crospovidone, copovidone, etc.), methylcellulose, hydroxypropyl methylcellulose, gelatinized starch (e.g. STARCH 1500® and STARCH 1500 LM® sold by Colorcon, Ltd. .), hydroxypropyl methylcellulose, microcrystalline cellulose (FMC Corporation, Marcus Hook, PA, USA), or mixtures thereof.

Fillers: talc, calcium carbonate (eg granules or powder), dibasic calcium phosphate, tribasic calcium phosphate, calcium sulfate (eg granules or powder), microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch , gelatinized starch, dextrose, fructose, honey, lactose anhydrous, lactose monohydrate, lactose and aspartame, lactose and cellulose, lactose and microcrystalline cellulose, maltodextrin, maltose, mannitol, microcrystalline cellulose &amp; guar gum, molasses, sucrose blends, raising agents such as agar-agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, potassium polacrilin, sodium carboxymethyl starch, potato or tapioca starch, other starches, gelatinized starch, clays, others algins, other celluloses, gums (like gellan), low-substituted hydroxypropyl cellulose, or mixtures thereof.

Lubricants: calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, sodium stearyl fumarate, vegetable fatty acid lubricant, talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethyl laurate, agar, siloid silica gel (AEROSIL 200, W.R. Grace Co., Baltimore, MD USA) , coagulated aerosol synthetic silica (Deaussa Co., Piano, TX USA), pyrogenic silica (CAB-O-SIL, Cabot Co., Boston, MA USA), or mixtures thereof.

Anti-caking agents: calcium silicate, magnesium silicate, silicon dioxide, colloidal silicon dioxide, talc or mixtures thereof.

Antimicrobial agents: benzalkonium chloride, benzethonium chloride, benzoic acid, benzyl alcohol, butylparaben, cetylpyridinium chloride, cresol, chlorobutanol, dehydroacetic acid, ethylparaben, methylparaben, phenol, phenylethyl alcohol, phenoxyethanol, phenylmercuric acetate, phenylmercuric nitrate, potassium sorbate, propylparaben, sodium benzoate, sodium dehydroacetate, sodium propionate, sorbic acid, thimerosal, thyme, or mixtures thereof.

Coating Agents: Sodium Carboxymethylcellulose, Cellulose Acetate Phthalate, Ethylcellulose, Gelatin, Pharmaceutical Glaze, Hydroxypropyl Cellulose, Hydroxypropyl Methylcellulose (Hypromellose), Hydroxypropyl Methylcellulose Phthalate, Methylcellulose, Polyethylene Glycol, Polyvinyl Acetate Phthalate, Shellac, Sucrose, Titanium Dioxide, Carnauba Wax, Microcrystalline Wax, Gellan Gum, maltodextrin, methacrylates, microcrystalline cellulose and carrageenan or mixtures thereof.

Dosage forms may also include other excipients and categories thereof, including, but not limited to, L-histidine, Pluronic®, Poloxamers (such as Lutrol® and Poloxamer 188), ascorbic acid, glutathione, penetration-enhancing agents (e.g., lipids, cholate sodium, acylcarnitine, salicylates, mixed bile salts, fatty acid micelles, chelating agents, fatty acid, surfactants, medium chain glycerides), protease inhibitors (eg, soybean trypsin inhibitor, organic acids), pH lowering agents, and absorption aids effective in providing bioavailability (including, but not limited to, the agents described in US 6086918 and US 5912014), creams and lotions (like maltodextrin and carrageenans); chewable tablet materials (like dextrose, fructose, lactose monohydrate, lactose and aspartame, lactose and cellulose, maltodextrin, maltose, mannitol, microcrystalline cellulose and guar gum, crystalline sorbitol); parenteral agents (like mannitol and povidone); plasticizers (like dibutyl sebacate, plasticizers for

- 23 040600 coatings, polyvinyl acetate phthalate); powdered lubricants (like glyceryl behenate); soft gelatin capsules (similar to a special solution of sorbitol); spheres to cover (similar to sugar spheres); spheroidizing agents (like glyceryl behenate and microcrystalline cellulose); suspending/gelling agents (like carrageenan, gellan gum, mannitol, microcrystalline cellulose, povidone, sodium carboxymethyl starch, xanthan gum); sweeteners (like aspartame, aspartame and lactose, dextrose, fructose, honey, maltodextrin, maltose, mannitol, molasses, crystalline sorbitol, sorbitol special solution, sucrose); wet granulating agents (like calcium carbonate, lactose anhydrous, lactose monohydrate, maltodextrin, mannitol, microcrystalline cellulose, povidone, starch), caramel, sodium carboxymethyl cellulose, cherry cream flavor and cherry flavor, anhydrous citric acid, citric acid, confectionery Sugar, D&C Red #33, D&C Yellow #10 Aluminum Varnish, Disodium Edetate, Ethyl Alcohol 15%, FD&C Yellow #6 Aluminum Varnish, FD&C Blue #1 Aluminum Varnish, FD&C Blue #1, FD&C Blue #2 Aluminum Varnish, FD&C Green No. 3, FD&C Red No. 40, FD&C Yellow No. 6 Aluminum Lacquer, FD&C Yellow No. 6, FD&C Yellow No. 10, Glycerol Palmitostearate, Glyceryl Monostearate, Indigo Carmine, Lecithin, Manitol, Methyl and Propyl Parabens, Monoammonium Glycyrrhizinate, Natural and Artificial Flavor orange, pharmaceutical glaze, poloxamer 188, polydextrose, polysorbate 20, polysorbate 80, polyvidone, gelatinized corn starch, gelatinized starch, iron oxide red, sodium saccharin, sodium carboxymethyl ether, sodium chloride, sodium citrate, sodium phosphate, strawberry flavor, synthetic iron oxide black, synthetic iron oxide red, titanium dioxide and white beeswax.

Solid oral dosage forms may optionally be treated with coating systems (e.g. Opadry® fx film coating system, e.g. Opadry® blue (OY-LS20921), Opadry® white (YS-2-7063), Opadry® white (YS-1- 7040) and black ink (S-1-8106).

The agents, either in their free form or in salt form, can be combined with a polymer such as polylactic glycolic acid (PLGA), poly(I)-lactic-glycolic-tartaric acid (P(I)LGT) (WO 01/ 12233), polyglycolic acid (US 3773919), polylactic acid (US 4767628), poly(ε-caprolactone) and poly(alkylene oxide) (US 20030068384) to create a sustained release dosage form. Such dosage forms can be used in implants that release a polypeptide or other agent over a period of several days, several weeks or several months depending on the polymer, polymer particle size and implant size (see, for example, US 6620422). Other sustained release dosage forms and polymers for use therein are described in EP 0467389 A2, wO 93/24150, US 5612052, WO 97/40085, WO 03/075887, WO 01/01964A2, US 5922356, WO 94/155587, WO 02/074247A2, WO 98/25642, US 5968895, US 6180608, US 20030171296, US 20020176841, US 5672659, US 5893985, US 5134122, US 5192741, US 5192741, US 4668506, US 4713244, US 5445832, US 4931279, US 5980945 , WO 02/058672, WO 97/26015, WO 97/04744 and US 20020019446. In such sustained release dosage forms, microparticles (Delie and Blanco-Prieto, 2005, Molecule, 10:65-80) of a polypeptide are combined with polymer microparticles. One or more sustained release implants may be placed in the large intestine, small intestine, or both. US 6011011 and WO 94/06452 describe a sustained release dosage form providing either polyethylene glycols (ie PEG 300 and PEG 400) or triacetin. WO 03/053401 describes a dosage form that can both improve bioavailability and provide controlled release of an agent within the gastrointestinal tract. Additional controlled release dosage forms are described in WO 02/38129, EP 326151, US 5236704, WO 02/30398, WO 98/13029; US 20030064105, US 20030138488A1, US 20030216307A1, US 6667060, WO 01/49249, WO 01/49311, WO 01/49249, WO 01/49311 and US 5877224, which may include controlled release formulations (including 1904 and the enteric coating described therein) and pH controlled coatings that provide delivery to the colon, including the pH controlled coatings described in US 4,910,021 and WO 9001,329. US 4,910,021 describes the use of a pH controlled material for coating capsules. WO9001329 describes the use of pH controlled coatings on acid-containing beads, where the acid in the base bead prolongs the dissolution of the pH controlled coating. US Pat. No. 5,175,003 discloses a blend of dual mechanism polymers consisting of pH controlled enteric materials and film-forming plasticizers capable of permeating the enteric material for use in drug delivery systems; pellets with a matrix consisting of a mixture of dual mechanism polymers impregnated with a drug and sometimes coating a pharmaceutically neutral core; membrane-coated pellets containing pellets with a matrix coated with a mixture of dual mechanism polymers from the same or a different composition; and a dosage form containing a matrix of pellets. The pellet matrix releases acid-soluble drugs by diffusion at acidic pH and by disintegration at pH levels nominally about 5.0 or higher.

In some embodiments, the compositions described herein are formulated in a pH dependent release form. Alternatively, such compositions are formulated in a form that releases the peptides in a specific region of the gastrointestinal (GI) tract (eg, duodenum, jejunum, ileum, terminal ileum, or ascending colon). The dosage form may contain an inert carrier coated with an enteric-coated composition that releases the peptides at a specific pH (such as pH 5 or pH 7). The preferred pH for release in the duodenum or jejunum is pH 4.5-5.5 or pH 5.5-6.5. The preferred pH for release in the ileum, terminal ileum or ascending colon is pH 5.5-6.5 or pH 6.5-7.5. Preferably the inert carrier is selected from mannitol, lactose, microcrystalline cellulose or starch.

The GCRA peptides described herein can be formulated with the pH triggered targeted controlled release system described in WO 04052339. The agents described herein can be formulated according to the procedure described in any of the WOs. 03105812 (extruded hydratable polymers); WO 0243767 (membrane translocators cleaved by enzymes); WO 03007913 and WO 03086297 (mucoadhesive systems); WO 02072075 (two-layer coated formulation comprising a pH lowering agent and an absorption aid); WO 04064769 (amidated polypeptides); WO 05063156 (suspension of solid lipids with pseudotropic and/or thixotropic properties after melting); WO 03035029 and WO 03035041 (disintegrating, gastric delay dosage forms); US 5007790 and US 5972389 (sustained release dosage forms); WO 04/112711 (oral compositions with prolonged action); WO 05027878, WO 02072033 and WO 02072034 (delayed release formulations with natural or synthetic resin); WO 05030182 (increasing rate controlled release dosage forms); WO 05048998 (microencapsulation system); US Pat. No. 5,952,314 (biopolymer); US 5108758 (matrix delivery with glassy amylose); US 5840860 (modified starch delivery). JP 10324642 (delivery system comprising chitosan and gastrointestinal resistant materials such as wheat gliadin or zein); US 5866619 and US 6368629 (polymer containing saccharide); US 6531152 (describes a drug delivery system containing a water-soluble core (Ca pectinate or other water-insoluble polymers) and an outer shell that is torn (eg hydrophobic Eudragrit polymer)); US 6234464; US 6403130 (coating with polymer containing casein and high methoxy pectin); WO 0174175 (Maillard reaction product); WO 05063206 (composition that increases the solubility); WO 04019872 (fusion carrier proteins).

Dosage forms with controlled release.

In one embodiment, the GCC agonist dosage form contains a targeting material that provides controlled (time dependent) release of the GCC agonist. Controlled release in this context includes delayed sustained release, delayed controlled release, delayed slow release, delayed extended release, delayed extended release and unexpected release or burst.

Preferably, the controlled release dosage form comprises a slowly disintegrating core containing a GCC agonist surrounded by a guide material. The guide material preferably contains at least one swellable polymer. Non-limiting examples of swellable polymers for use in the controlled release dosage form of the invention include acrylic copolymers such as EUDRAGIT RL, EUDRAGIT RS or EUDRAGIT NE; polyvinyl acetate, such as KOLLICOAT SR 30D; and cellulose derivatives such as ethylcellulose or cellulose acetate, such as SURELEASE and AQUACOAT ECD. In a preferred embodiment, the guide material comprises one or more of EUDRAGIT RL, EUDRAGIT RS, or EUDRAGIT NE to provide timed release of the GCC agonist by pH independent swelling. In a separate embodiment, the guide material contains EUDRAGIT RL:RS (2:8) and an outer coating containing EUDRAGIT FS.

Additional non-limiting examples of swellable polymers that can be used in sustained release dosage forms of the invention include poly(hydroxyalkyl methacrylate) having a molecular weight of 30,000 to 5,000,000; kappa-carrageenan; polyvinylpyrrolidone having a molecular weight of 10,000 to 360,000; anionic and cationic hydrogels; polyelectrolyte complexes; poly(vinyl alcohol) having low amounts of acetate cross-linked with glyoxal, formaldehyde or glutaraldehyde and having a polymerization degree of 200 to 30,000; a mixture containing methylcellulose, cross-linked agar and carboxymethylcellulose; water-insoluble, water-swellable copolymer obtained by forming a dispersion of finely divided maleic anhydride with styrene, ethylene, propylene, butylene

- 25 040600 or isobutylene; water-swellable polymers of N-vinyl lactams; polysaccharide, water-swellable gums, high viscosity hydroxypropyl methylcellulose, and/or mixtures thereof. In some embodiments, the swellable polymer is selected from the group consisting of calcium pectinate, a cross-linked polysaccharide, water-insoluble starch, microcrystalline cellulose, water-insoluble cross-linked peptide, water-insoluble cross-linked protein, water-insoluble cross-linked gelatin, water-insoluble cross-linked hydrolyzed gelatin, water-insoluble cross-linked collagen, modified cellulose and cross-linked polyacrylic acid. Non-limiting examples of the cross-linked polysaccharide include insoluble metal salts or cross-linked derivatives of alginate, pectin, xanthan gum, guar gum, gum tragacanth and locust bean gum, carrageenan, their metal salts, and their covalent cross-linked derivatives. Non-limiting examples of modified cellulose include cross-linked derivatives of hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, methylcellulose, carboxymethylcellulose, and metal salts of carboxymethylcellulose.

In some embodiments, the implementation of the swellable core also contains a capillary agent, such as silicon dioxide. The capillary agent may also be selected from a disintegrant such as microcrystalline cellulose to enhance the rate of water absorption. Other suitable capillary agents include, but are not limited to, kaolin, titanium dioxide, fumed silica, alumina, niacinamide, sodium lauryl sulfate, low molecular weight polyvinylpyrrolidone, m-pyrol, bentonide, magnesium aluminum silicate, polyester, polyethylene, and mixtures thereof.

In some embodiments, the guide material, which may comprise a portion of the core and/or form one or more layers covering the core, optionally further comprises at least one of a lubricant, a flow aid, a plasticizer, a detackifier, a surfactant, a wetting agent, a suspending agent and a dispersing agent.

In some embodiments, the guide material comprises a water insoluble polymer and a blowing agent. Non-limiting examples of blowing agents include sucrose, sodium chloride, potassium chloride, polyvinylpyrrolidone and/or polyethylene glycol, water soluble organic acids, sugars and sugar alcohol. In some embodiments, the implementation of the blowing agent forms part of the outer layer or coating. In other embodiments, the pore-forming agent is unevenly distributed throughout the water-insoluble polymer.

In one embodiment, the guide material comprises a press-on coating.

Non-limiting examples of materials that can be used as a compression coating include a gum selected from the group consisting of xanthan gum, locust bean gum, galactans, mannans, alginates, karaya gum, pectin, agar, tragacanth, acacia gum, carrageenan, tragacanth, chitosan, agar, alginic acid, acacia catechu hydrocolloids, salai guggal gum, Indian bdelia gum, copaiba gum, asafoetida, cambi gum, Enterolobium cyclocarpum gum, mastic gum, benzoin gum, sandarac, kabira gum, Butea frondosa gum (plant gum) forest fire), myrrh, konjac mannana, guar gum, welan gum, gellan gum, tara gum, locust bean gum, carrageenan gum, glucomannan, galactan gum, sodium alginate, tragacanth, chitosan, xanthan gum, deacetylated xanthan gum, pectin, sodium polypectate, gluten, karaya gum, tamarind gum, ghatti gum, acaroid/Yacca/red gum, dammar gum, juniper gum, ester gum, ipil-ipil seed gum, talha gum (Acacia seyal) and cultivated plant cell gums, including gums of plant genera: acacia, actinidia, aptenia, carbobrotus, chickorium , cucumis, glycine, hibiscus, hordeum, letuca, lycopersicon, malus, medicago, mesembryanthemum, oryza, panicum, phalaris, phleum, poliathus, polycarbophyl, sida, solanum, trifolium, trigonella, Afzelia africana seed gum, Treculia africana gum, detarium gum , cassia gum, carob gum, Prosopis africana gum, Colocassia esulenta gum, Hakea gibbosa gum, khaya gum, scleroglucan and zea, and mixtures of any of these.

In some embodiments, the guide material further comprises a plasticizer, a stiffening agent, a wetting agent, a suspending agent, a dispersing agent, or a combination thereof. Non-limiting examples of a plasticizer include dibutyl sebacate, polyethylene glycol and polypropylene glycol, dibutyl phthalate, diethyl phthalate, triethyl citrate, tributyl citrate, acetylated monoglyceride, acetyl tributyl citrate, triacetin, dimethyl phthalate, benzyl benzoate, butyl and/or glycol fatty acid esters, refined mineral oils, oleic acid, castor oil, corn oil, camphor, glycerin and sorbitol, or a combination thereof. In one embodiment, the hardening agent comprises cetyl alcohol. Non-limiting examples of wetting agents include poloxamer, polyoxyethylene ethers, polyoxyethylene sorbitan fatty acid esters, polyoxymethylene stearate, sodium lauryl sulfate, sorbitol fatty acid esters, benzalkonium chloride, poly

- 26 040600 ethoxylated castor oil and sodium docusate. Non-limiting examples of suspending agents include alginic acid, bentonide, carbomer, carboxymethylcellulose, calcium carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, microcrystalline cellulose, colloidal silicon dioxide, dextrin, gelatin, guar gum, xanthan gum, kaolin, magnesium aluminum silicate, maltitol, medium chain triglycerides, methylcellulose, polyoxyethylene sorbitol fatty acid esters, polyvinyl pyrrolidinone, propylene glycol alginate, sodium alginate, sorbitol fatty acid esters and gum tragacanth. Non-limiting examples of dispersing agents include poloxamer, polyoxyethylene sorbitol fatty acid esters, and sorbitol fatty acid esters.

In some embodiments, the targeted release dosage form further comprises an outer enteric coating over the targeted release material. Preferably the enteric coating is selected from the group consisting of cellulose acetate phthalate, hydroxypropyl methylcellulose acetate succinate, EUDRAGIT L100 and EUDRAGIT L30D-55.

Explosive dosage form.

In one embodiment, the GCC agonist dosage form is a time-delayed dosage form capable of rapid explosive release of the GCC agonist in the colon or small intestine (explosive dosage form). The dosage form contains a core and an outer layer. The core contains at least one GCC agonist and at least one controlled burst agent. In some embodiments, the core further comprises at least one disintegrant selected from the group consisting of croscarmellose sodium, crospovidone (crosslinked PVP), sodium carboxymethyl starch (sodium starch glycolate), crosslinked sodium carboxymethylcellulose (croscarmellose), gelatinized starch ( starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethylcellulose and magnesium aluminosilicate, or combinations thereof. In other embodiments, the core further comprises at least one of an absorbent, a binder, a hardening agent, a buffering agent, a filler, a flow control agent, a lubricant, a mutually reinforcing agent, a chelating agent, an antioxidant, a stabilizer, and a preservative. Optionally, the core also contains one or more other excipients.

The controlled burst agent in the core preferably contains a water insoluble polymer to control the rate of penetration of water into the core and increase the internal pressure (osmotic pressure) within the core. Such a controlled explosion agent is preferably capable of swelling upon contact with a liquid. Non-limiting examples of suitable water-insoluble polymers include cross-linked polysaccharide, water-insoluble starch, microcrystalline cellulose, water-insoluble cross-linked peptide, water-insoluble cross-linked protein, water-insoluble cross-linked gelatin, water-insoluble cross-linked hydrolyzed gelatin, water-insoluble cross-linked collagen, modified cellulose and cross-linked cross-linked polyacrylic acid. In one embodiment, the water-insoluble polymer is a cross-linked polysaccharide selected from the group consisting of insoluble metal salts or cross-linked derivatives of alginate, pectin, xanthan gum, guar gum, tragacanth gum and locust bean gum, carrageenan gum, salts thereof. metals and their covalent cross-linked derivatives. In one embodiment, the water-insoluble polymer is a modified cellulose selected from the group consisting of cross-linked derivatives of hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, methylcellulose, carboxymethylcellulose, and metal salts of carboxymethylcellulose. In yet another embodiment, the water insoluble polymer is selected from calcium pectinate, microcrystalline cellulose, or a combination thereof.

The outer layer contains a water-insoluble hydrophobic carrier and a pore-forming agent consisting of a water-insoluble hydrophilic special substance.

The blowing agent is a permeable agent that allows liquid to enter the core. Optionally, the outer layer further comprises at least one of a wetting agent, a suspending agent, a dispersing agent, a hardening agent, and a plasticizer.

In some embodiments, the water-insoluble hydrophobic carrier is selected from the group consisting of a dimethylaminoethyl acrylate/ethyl methacrylate copolymer, wherein the copolymer is based on esters of acrylic and methacrylic acid with a low content of quaternary ammonium groups, wherein the molar ratio of ammonium groups to the remaining neutral esters of (meth)acrylic of acid is approximately 1:20, a polymer corresponding to USP/NF Ammonium Methacrylate Copolymer Type A, an ethyl methacrylate/chlorotrimethylammonium methyl methacrylate copolymer, an acrylic methacrylic acid ester copolymer with a low content of quaternary ammonium groups, with a molar ratio of ammonium groups and the rest

- 27 040600 neutral esters of (meth)acrylic acid is 1:40, a polymer corresponding to USP/NF Ammonium-Methacrylate Copolymer Type B, a copolymer of dimethylaminoethyl methacrylate/methyl methacrylate and butyl methacrylate, a copolymer based on neutral esters of methacrylic acid and esters of dimethylaminoethyl methacrylate, wherein the polymer is cationic in the presence of acids, ethyl acrylate and a copolymer of methyl acrylate/ethyl methacrylate and methyl methyl acrylate, the copolymer being a neutral copolymer based on neutral esters of methacrylic acid and acrylic acid, ethyl cellulose, shellac, zein and waxes.

In some embodiments, the water-insoluble particulate material is a hydrophilic as well as water-insoluble polymer, preferably selected from the group consisting of a water-insoluble cross-linked polysaccharide, a water-insoluble cross-linked protein, a water-insoluble cross-linked peptide, a water-insoluble cross-linked gelatin, a water-insoluble cross-linked hydrolyzed gelatin, water-insoluble cross-linked collagen, water-insoluble cross-linked polyacrylic acid, water-insoluble cross-linked cellulose derivatives, water-insoluble cross-linked polyvinylpyrrolidone, microcrystalline cellulose, insoluble starch, microcrystalline starch, and combinations thereof. Most preferably, the water insoluble particulate material is microcrystalline cellulose.

In some embodiments, the explosive dosage form further comprises an enteric coating on the outer layer. The enteric coating is preferably selected from the group consisting of cellulose acetate phthalate, hydroxypropyl methylcellulose acetate succinate and a EUDRAGIT polymer such as EUDRAGIT L100 or EUDRAGIT L30D-55.

Biodegradable dosage forms.

In one embodiment, the GCC agonist dosage form contains a natural or synthetic polymer that is degradable by at least one bacterial colonic enzyme. Preferably, the GCC agonist is embedded in a polymeric matrix. Non-limiting examples of such polymers include polymers of polysaccharides such as amylase, chitosan, chondroitin sulfate, cyclodextrin, dextran, guar gum, pectin, and xylan. Preferably, the natural or synthetic polymer is gelled or cross-linked with a cation such as a zinc cation, for example from zinc sulfate, zinc chloride or zinc acetate. The dosage form is preferably in the form of ionically cross-linked beads which are subsequently enteric coated. The enteric coating may comprise any suitable enteric coating material such as hydroxypropyl methylcellulose phthalate, polyvinyl acetate phthalate, cellulose acetate phthalate, hydroxypropyl methyl cellulose acetate succinate, alginic acid and sodium alginate, or EUDRAGIT polymer.

In yet another embodiment, the GCC agonist dosage form comprises a GCC agonist covalently conjugated to a carrier molecule such that the covalent bond between the GCC agonist and the carrier in the stomach and small intestine is stable but labile in the lower gastrointestinal tract, especially in large intestine. A GCC agonist covalently linked to a carrier molecule is referred to as a GCC prodrug. In some embodiments, the GCC prodrug comprises a GCC agonist covalently conjugated to the carrier molecule via an azo bond or a glycosidic bond. In other embodiments, the GCC prodrug contains a glucuronide, a cyclodextrin, a dextran ester, or a polar amino acid. In some embodiments, the GCC prodrug is a polymeric prodrug. In one embodiment, the polymeric prodrug contains polyamides containing azo groups.

Dosage.

The GCRA peptides described herein can be formulated using gastrointestinal retention system (GIRES; Merrion Pharmaceuticals) technology. GIRES contains a controlled release formulation within an inflatable bag that is placed in a drug capsule for oral administration. Upon dissolution of the capsule, the gas generating system inflates the bag in the stomach, where it remains for 16-24 hours, all the while releasing the substances described herein.

The GCRA peptides described herein can be formulated in an osmotic device, including the osmotic devices disclosed in US 4,503,030, US 5,609,590, and US 5,358,502. certain pH. More specifically, the invention relates to an osmotic device comprising a wall formed from a semi-permeable pH sensitive composition that surrounds a compartment containing a drug, with a passage through the wall connecting the outer part of the device to the compartment. The device delivers drug at a controlled rate to an area of the gastrointestinal tract having a pH of less than 3.5, and the device self-destructs and releases all of its drug to an area of the gastrointestinal tract having a pH of more than 3.5, thereby providing a total availability for drug absorption. U.S. Patent Nos. 5,609,590 and 5,358,502 disclose

- 28 040600 osmotic explosive device for dispersing a beneficial substance into an aqueous environment. The device contains a beneficial substance and an osmotic agent, surrounded by at least partially semi-permeable membrane. The beneficial substance can also act as an osmotic agent. The semi-permeable membrane is permeable to water and substantially impermeable to the beneficial agent and the osmotic agent. The initiating agent is attached to a semi-permeable membrane (eg, connects the two halves of the capsule).

The initiating agent is activated by a pH of 3 to 9 and initiates a possible, but unexpected, delivery of the beneficial agent. These devices allow pH triggered release of the core from the beneficial agent as a bolus via an osmotic burst.

Dosage levels of the active ingredients in the pharmaceutical composition may also be varied in order to achieve a transient or steady state concentration of the compound in the patient, especially at and around the site of inflammation or disease area, and to produce the desired response. It is well within the skill in the art to start doses of the compound at levels lower than those required to achieve the desired effect and gradually increase the dosage until the desired effect is achieved. It should be understood that the specific dosage level for any individual patient will depend on a number of factors including body weight, general health, nutrition, natural course of the disease, route and schedule of administration, combination with one or more other drugs, and severity of the disease.

An effective dose of the composition will typically be between about 1 µg and about 10 mg per kilogram of body weight, preferably between about 10 µg and 5 mg of the compound per kilogram of body weight. Dosage adjustments will be made using methods that are generally accepted in the art and will be based on the particular formulation being used and clinical considerations.

The guanylate cyclase receptor agonists used in the methods described above can be administered orally, systemically, or topically. Dosage forms include preparations for inhalation or injection, solutions, suspensions, emulsions, tablets, capsules, topical balms and lotions, transdermal compositions, other known peptide dosage forms, and pegylated peptide analogs. Agonists can be administered either as the sole active agent or in combination with other drugs, such as a cGMP-dependent phosphodiesterase inhibitor and an anti-inflammatory agent. In all cases, the additional drugs must be administered at a dosage that is therapeutically effective, using the current state of the art as guidance. Drugs can be administered in a single composition or sequentially.

Dosage levels of a GCR agonist for use in the methods of this invention are typically from about 0.001 mg to about 10,000 mg per day, preferably from about 0.005 mg to about 1,000 mg per day. For example, an effective dosage of peptide-GCRA for use in the methods of this invention is about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, approximately 0.9, approximately 1.0, approximately 1.5, approximately 2.0, approximately 2.5, approximately 3.0, approximately 3.5, approximately 4.0, approximately 4.5, approximately 5, 0, approximately 5.5, approximately 6.0, approximately 6.5, approximately 7.0, approximately 7.5, approximately 8.0, approximately 8.5, approximately 9.0, approximately 9.5, approximately 10, approximately 11 , about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 mg per day, or optionally twice daily. Preferably, the GCRA peptide is given after a meal (ie, 30 minutes later). In some embodiments, a second agent is administered that is used to treat a lipid metabolic disorder, a biliary disorder, a cardiovascular disease, obesity, or an endocrine disorder. Suitable second agents are described in this document. In some aspects, the second agent is administered at less than standard doses to treat a particular disorder because the GCRA peptide acts in synergy with the second agent. For example, about 2.5, about 5, about 7.5, or about 10 mg of Liptor is given twice a day after meals (ie, 30 minutes later). Depending on the mg/kg daily dose given either in single or divided doses, dosages typically range from about 0.001/75 mg/kg to about 10,000/75 mg/kg, preferably from about 0.005/75 mg/kg to about 1,000/75 mg/kg.

The total daily dose of each inhibitor may be administered to the patient in a single dose or in multiple sub-doses. Generally, subdoses can be administered two to six times a day, preferably two to four times a day, and even more preferably two to three times a day. Doses may be in an immediate release dosage form or in a sustained release dosage form effective enough to obtain the desired control over the course of the disease state.

A drug regimen, on the one hand, to prevent, treat, alleviate or alleviate a pathological condition or disorder, or, on the other hand, to protect against

- 29 040600 medical condition or treatment of a pathological condition with combinations and compositions of the present invention is selected in accordance with a number of factors. These factors include, but are not limited to, constitution, age, body weight, gender, nutrition and pathological condition of the patient, severity of disease, route of administration, pharmacological aspects such as action, efficacy, pharmacokinetic and toxicological profiles of the specific inhibitors used, whether a delivery system is used drug or whether inhibitors are administered with other active ingredients. Thus, the actual reception scheme used may vary widely and therefore deviate from the preferred reception scheme set forth above.

Examples

Example 1 Oral treatment with SP-333, a uroguaniline analog, alleviates morphine- and methadone-induced constipation through activation of guanylate cyclase-C and cystic fibrosis transmembrane conductance regulator in rats.

Opioids are widely used to treat chronic pain, but their use is often associated with severe constipation in people for whom standard laxatives are not very effective. We would like to believe that this side effect occurs due to a decrease in the secretion of fluids, affecting the contractility of the gastrointestinal (GI) tract and defecation (BM). The mechanism through which opioid-induced stool impairment occurs is shown in FIG. 2. Thus, there is a need for a safe, orally administered pharmacological agent that can relieve constipation induced by a broad class of opioids.

SP-333, a uroguaniline analog, activates GC-C to stimulate the production of cyclic GMP, which in turn activates the cystic fibrosis transmembrane conductance regulator (CFTR), an apical chloride channel responsible for the efflux of chloride ions from enterocytes lining the gastrointestinal ( GI) tract, resulting in a net efflux of water into the intestinal lumen to facilitate defecation and GI contractility (Shailubhai, 2002; Forte, 2004; Basu et al., 2010; Steinbrecher, 2014).

This study demonstrates the potential of SP-333 as an oral therapeutic drug for the treatment of opioid-induced constipation (OIC). Treatment with SP-333 causes a concentration-dependent increase in short-circuit currents (Isc) through the T-84 cell monolayer and rat jejunal tissue, even in the presence of morphine or methadone. This increase in short circuit current appears to be mediated through the GC-C/cGMP activation mechanism of the G-II kinase protein and the cystic fibrosis transmembrane conductance regulator. In rat studies, daily intraperitoneal administration of methadone for seven days created a significant delay in GI passage, resulting in a marked decrease in daily fecal excretion. Orally administered SP-333 completely reversed GI transit delay induced by morphine or methadone. Oral treatment with SP-333 (2.5 and 5 mg/kg/day) not only normalized gastrointestinal transit, but also partially restored fecal excretion.

Materials and methods.

Cell culture.

T84, a human cancer cell line, was purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA). Cells were cultured in DMEM/F-12 medium (Fisher Scientific, Pittsburgh, PA) containing 10% fetal bovine serum (Atlanta Biologicals, Flowery Branch, GA), 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA), and 1% glutamax. (Invitrogen, Carlsbad, CA) as described (Shailubhai et al., 2000). For Ussing chamber experiments, cells were grown on Snapwell inserts of 12 mm polyester permeable membrane (pore size 0.4 μm) at a seeding density of ~150,000 cells/insert (CLS 3801, Sigma, Saint Louis, MO) and incubated at 37°C in a humidified atmosphere containing 5% CO ₂ .

Animals.

All study manipulations were performed according to the study summary guidelines approved by the Institutional Animal Care and Use Committee of MB Research Laboratories (Spinnerstown, PA) or Lampire Biological Laboratories (Pipersville, PA). Female Sprague-Dawley [Crl:CD(SD)] rats, seven to eight weeks old, weighing between ~180-210 g, were obtained from Charles River Laboratories and allowed to acclimate for a week. Animals were kept on a 12-hour light-dark cycle. To collect tissue from the small intestine and assess gastrointestinal transit, the rats were fasted for at least 18 h prior to the experiment with free access to water.

Rat tissue collection.

Rats were euthanized by CO2 inhalation. A median abdominal incision was made. A proximal 5 cm section of jejunum 1 cm distal to the ligament of Treitz was identified, excised and transferred to ice-cold RPMI containing indomethacin (10 μm). After 10 min, the tissue was transferred to fresh ice-cold RPMI without indomethacin. The tissue was pinned onto a dissection plate and dissected along the mesenteric margin with the apical end pointing

- 30 040600 up, and placed on the Ussing chamber slides as described below.

Measurement of short circuit currents.

Isc measurements were performed on an EasyMount Ussing chamber system (4 chambers) with VCCMC8 multichannel current-voltage (IV) terminals (Physiologic Instruments, San Diego, CA). Trensepithelial resistance (TER) across a monolayer of cells cultured on permeable membranes was routinely monitored using an EVOM epithelial volt-ohmmeter (EMD Millipore, MA). A continuous layer of T-84 cells with TER >1000 ohm-cm ² was used. Cells and tissues were placed on specific sliders (slit area 1.12 cm ² for cells and 0.5 cm ² for tissue) provided by Physiologic Instruments and placed in a Ussing chamber. The chambers were immersed for a long time in Krebs-Ringer's buffer solution. The solution washing the basolateral surface contained (in mM) 115 NaCl, 25 NaHCO3, 3.3 KH2PO4, 0.8 K2HPO4, 1.12 MgCl2, 1.2 CaCl2, and 10 HEPES (Cuppoletti et al., 2004). In order to investigate the effect of SP-333 or specific inhibitors on chloride channels on the apical membrane, a chloride gradient was generated from the basolateral to apical surface by displacing NaCI in a buffer solution in contact with the apical membrane with an equal concentration of sodium gluconate. Since gluconate chelates Ca2 ⁺ ions, the CaCl2 concentration increased to 4 mM (Bijvelds et al., 2009). The buffer solution was continuously saturated with carbon (a mixture of 95% O ₂ and 5% CO ₂ ) and the temperature of 37°C was kept constant with a heating block. The lemmas were connected to the Acquire & Analyze software (Physiologic Instruments) to automatically collect data from all four chambers. Ag/AgCl control electrodes were used to measure transepithelial voltage and passing current.

To evaluate SP-333-stimulated short circuit current through a monolayer of T-84 cells (n=4 transvel/concentration) and rat jejunal tissue (n=4 drugs/concentration), indicated amounts of agonist were added to the apical buffer solution after stabilization of the initial state and peak activity was recorded and plotted as Isc (μA/cm ² )±standard error of the mean. The effect of morphine or methadone on SP-333 stimulated short circuit current was determined by pretreatment of cells (n=3 transwell/opioid concentration) and rat jejunum (n=4-15 drugs/opioid concentration) on the apical side with opioid for 5 min ( Cuppoletti et al., 2013). Subsequently, 1 μM SP-333 was added to the buffer solution on the apical side, the peak activity of stimulated currents was measured and expressed as chloride currents (Isc) (relative to the vehicle control group) ± standard error of the mean. To assess the role played by PKA and PKG-II and to determine the relative contributions of CFTR and CIC-2 in relation to agonist-mediated release of Cl ions, experiments were performed in the presence and absence of kinase and CT channel inhibitors. The inhibitors used were CdCl ₂ (CIC-2 inhibitor; Sigma-Aldrich; St Louis, MO), CFTR _inh 172 (CFTR inhibitor; Santa Cruz Biotech; Dallas, Texas), KT5823 (PKG inhibitor; R&D Systems, Minneapolis, MN), mPKI (PKA inhibitor; EMD Millipore; Billerica, MA). Prior to addition of 1 mM SP-333, T-84 cells (n=4 transwells/condition) and jejunal tissue (n=4-6 drugs/condition) were pretreated for 5 minutes with each inhibitor at the indicated concentration. The peak current obtained after the addition of SP-333 was recorded and calculated in terms of percentage values of relative activity ± standard error of the mean.

Effect of SP-333 on opioid-induced gastrointestinal transit delay in rats.

The ability of opioids to delay gastrointestinal transit in rats was investigated either by administration of 0 (vehicle), 0.25, 1 and 2.5 mg/kg morphine or 0 (vehicle), 0.25, 2.5 and 5 mg/kg methadone (volume dose 1.5 ml/kg) by intraperitoneal injection. Water served as a control solvent. 10 min later, 2 ml of an activated charcoal feed consisting of 10% activated charcoal (Sigma-Aldrich, St. Louis, MO) and 10% acacia gum (Sigma-Aldrich, St. Louis, MO) in water was administered via oral feeding through a stomach tube. Ten minutes after the charcoal diet, the rats were euthanized by intraperitoneal injection of 150 mg/kg Beuthanasia-D solution (sodium pentobarbital, 390 mg/ml). The small intestine was removed and the total length from the pyloric sphincter to the ileocecal junction was recorded, as well as the distance traveled by the leading edge of the coal. Intestinal transit was determined by calculating the ratio (expressed as a percentage) of the distance between the pyloric sphincter and the leading edge of the charcoal to the distance between the pyloric sphincter and the ileocecal junction.

To evaluate the ability of SP-333 to reverse morphine-induced gastrointestinal transit delay, animals were administered 2.5 mg/kg morphine via intraperitoneal injection immediately after oral gavage 0 (vehicle), 0.5, 2.5, 5 and 50 mg/kg SP-333 (volume dose 1.5 ml/kg). Water served as a solvent control. After 10 minutes, the charcoal diet was administered, and 10 minutes after the activated charcoal diet, the rats were euthanized and intestinal transit was calculated as described above.

To evaluate the ability of SP-333 to reverse methadone-induced gastrointestinal transit delay, animals were administered 2.5 mg/kg methadone via intraperitoneal injection. 10 minutes later, immediately after oral administration through a gastric tube of 2 ml of nutrition,

- 31 040600 containing charcoal, orally via gavage was administered 0 (vehicle), 0.1, 0.5, 2.5, 5 and 50 mg/kg

SP-333 (volume dose 1.5 ml/kg). 10 minutes after the activated charcoal diet, the rats were euthanized and intestinal transit was calculated as described above.

The ability of SP-333 to attenuate methadone-induced gastrointestinal transit delay was also evaluated in a similar manner. Female Sprague-Dawley rats 7 to 8 weeks old (n=7/group) received 16 doses of methadone (once a day; 7 doses of 5 mg/kg and 9 doses of 2.5 mg/kg) by intraperitoneal injection. ). Ten minutes after each dose of methadone, each group received oral gavage feeding of the vehicle, 2.5, and 5 mg/kg SP-333. At the end of the drug regimen, the animals were fasted overnight and gastrointestinal transit was assessed as described above.

Effect of SP-333 on methadone-induced constipation in rats.

To identify the dose of methadone that induces constipation in rats, female Sprague Dawley rats aged seven to eight weeks (n=6/group) were administered 0 (vehicle), 2.5, 5, and 7.5 mg/kg methadone ( 1.5 ml/kg volume dose) by intraperitoneal injection once a day for 5 days. Fecal lumps were collected at 4 h intervals during the light cycle and the average number of lumps for each group during the study was recorded after administration of the daily dose of methadone.

To assess the ability of SP-333 to reduce methadone-induced constipation to induce constipation, Sprague-Dawley female CD rats aged 7 to 8 weeks (n=7/group) were administered 5 mg/kg methadone (or vehicle) every day for 7 days by intraperitoneal injection, every day 10 min after the intraperitoneal dose, oral was administered through a gastric tube 2.5 and 5 mg/kg of SP-333 or vehicle (buffer). Fecal lumps were collected at 4-hour intervals during the light cycle and the average number of lumps for each group during the study was recorded after administration of the daily dose of vehicle, methadone, or a combination of methadone and SP-333.

Statistical analysis.

GraphPad Prism (version 6.01) was used to calculate summary statistics and inferential tests. Data, expressed as mean ± standard error of the mean and differences between the control group and the treatment group, were assessed for significance by a two-tailed Student's t-test.

Results.

PKG-II- and CFTR-channel-dependent stimulation of CI' currents by SP-333 in T-84 cells and rat jejunum. SP-333 stimulates ^CI currents (expressed as increased short circuit current) in T-84 cells and rat jejunum. The results are shown in FIG. 3. Incorporation of SP-333 (0.01-10 µm) into a buffer solution in contact with the mucosal surface of T-84 cells (Fig. 3A) or rat jejunum (Fig. 3B) resulted in a concentration-dependent increase in short circuit current, approaching saturation at 10 µm in both cases. The total short circuit current from the jejunal tissues was significantly higher than that observed for T-84 cells. However, the concentration of SP-333 that induced a 50% increase in short circuit current in a monolayer of T-84 cells (EC50=1.69x10' ⁷ M) or jejunal tissue (EC50=2.99x10' ⁷ M) was comparable.

In T-84 cells, 80% inhibition of SP-333-stimulated short circuit current was observed in the presence of 10 μM CFTRj _nh 172 compared to the inhibition observed alone with 1 μM SP-333 (FIG. 4A). The relative short circuit current in the presence of the PKG-II inhibitor (4 μm) was also suppressed by approximately 35% (FIG. 4A). No marked decrease in stimulated short circuit current was observed in the presence of the CIC-2 inhibitor, CdCl ₂ (300 μm) and 3.2 μM protein kinase inhibitor A (mPKI-) (FIG. 4A). A similar pattern was observed for rat jejunum, although the amount of inhibition with equal amounts of CFTR _inh 172 was less compared to that found in T-84 cells (FIG. 4B). Taken together, the results suggest that SP-333 mediated increases in short circuit current in the cell system model as well as in rat tissues require activated PKG-II and CFTR channels.

Effect of morphine and methadone on SP-333-stimulated ^CI currents. The effect of selected concentrations of morphine and methadone on SP-333-stimulated short circuit current was evaluated in T-84 cells and rat jejunum. The results are shown in FIG. 5 (morphine) and FIG. 6 (methadone).

Apical membrane pretreatment with 5 μM morphine had negligible effect on 1 μM SP-333-stimulated short circuit current in T-84 cells and rat jejunal tissues (FIG. 5). Similarly, T-84 cells and rat jejunum pretreated with 5 μM methadone had no effect on SP-333 mediated currents (FIGS. 6A and 6B).

Effect of oral administration of SP-333 on morphine-induced gastrointestinal transit delay in rats. A dose-dependent increase in small intestinal transit was observed after a single oral gavage dose of SP-333 (5.6, 13, 23% at 0.05, 0.5 and 5 mg/kg, respectively; see Fig. 7) compared with media control if treatment alone with SP-333 is expected to cause an increase in gastrointestinal transit in animals not previously used in experiments with opioids (Joshi et al., 2014). Intraperitoneal (IP) administration of morphine significantly reduced gastrointestinal transit in rats (FIG. 8A). Compared to medium controls, gastrointestinal transit after a single dose of morphine was slowed by 52%, 56%, and 56% at 0.25, 1.0, and

2.5 mg/kg opioid, respectively.

To evaluate the ability of SP-333 to reduce morphine-induced GI transit delay, animals were administered 2.5 mg/kg morphine via intraperitoneal injection directly followed by oral gavage 0 (vehicle), 0.5, 2.5, 5 and 50 mg/kg SP-333. Animals were sacrificed 10 min later and gastrointestinal transit calculated as described in Materials and Methods. Morphine significantly reduced gastrointestinal transit (23% vs. 53% with media treatment; p<0.0001) (FIG. 8B). SP-333 produced a dose-dependent improvement in intestinal transit when rats were treated with morphine: 27%, 37%, 46%, and 48% at 0.5, 2.5, 5, and 50 mg/kg, respectively (P<0.0001 at all doses for except for 0.5 mg/kg compared to morphine alone). The results suggest that a dose of 5 mg/kg of SP-333 is sufficient to reduce the effect of morphine on gastrointestinal transit. The mean gastrointestinal transit ± standard error of the mean is depicted in FIG. 8B.

Effect of oral administration of SP-333 on methadone-induced gastrointestinal transit delay in rats.

Intraperitoneal administration of methadone significantly reduced gastrointestinal transit in rats. A methadone dose of 2.5 mg/kg was sufficient to significantly reduce gastrointestinal transit. Additionally, the results were comparable to those observed with morphine at 2.5 mg/kg (49% and 56% vs. corresponding medium controls for methadone and morphine, respectively). The mean gastrointestinal transit ± standard error of the mean is depicted in FIG. 9A.

To assess the ability of SP-333 to reduce methadone-induced delayed gastrointestinal transit, animals were administered 2.5 mg/kg methadone via intraperitoneal injection. 10 min later, 0 (vehicle), 0.5, 2.5, 5, and 50 mg/kg SP-333 were orally administered via gavage. Animals were sacrificed 10 min later and gastrointestinal transit calculated as described in Materials and Methods. Methadone significantly reduced gastrointestinal transit (28% vs. 55% with medium treatment; P<0.0001). As shown in FIG. 9B, administration of SP-333 produced a dose-dependent improvement in intestinal transit in methadone-treated rats: 40%, 39%, 44%, and 52% at 0.5, 2.5, 5, and 50 mg/kg, respectively (P<0.01 at all doses). These data confirm that a dose of 5 mg/kg of SP-333 is sufficient to reduce the effect of methadone on gastrointestinal transit. The mean gastrointestinal transit ± standard error of the mean is depicted in FIG. 9B.

Effect of oral administration of SP-333 on constipation induced in rats by repeated administration of methadone.

Constipation was induced in rats by intraperitoneal administration of 2.5, 5, and 7.5 mg/kg methadone once a day for a period of 5 days. The average number of fecal boluses collected over a 4-hour period during the light cycle was significantly lower in the methadone treated group compared to the vehicle control group; 29, 77 and 54%, lower at 2.5, 5 and 7.5 mg/kg, respectively (P<0.01 at all doses) (FIG. 10A). The most pronounced effect of methadone on fecal excretion was observed at a dose of 5 mg/kg. To evaluate the ability of SP333 to reduce methadone-induced constipation, animals were administered 5.0 mg/kg methadone via intraperitoneal injection once a day for a period of 7 days. 10 minutes after opioid administration, 0 (vehicle), 2.5 and 5 mg/kg of SP-333 was orally administered via gavage every day and fecal excretion was monitored over a 4 hour time period during the light cycle. As shown in FIG. 10B, a significant reduction in mean fecal lumps was observed in animals treated with methadone alone (82% less versus medium control). The average number of lumps at all doses of SP-333 was higher compared to the methadone group. A three-fold increase in the number of lumps was observed at 2.5 mg/kg SP-333 compared to the number of lumps observed for the methadone-administered group that did not receive SP-333, demonstrating that SP333 relieves constipation in rats after repeated administration of methadone. Additionally, administration of SP-333 attenuated the delay in gastrointestinal transit induced by repeated dosing of methadone (FIG. 11).

Discussion.

Ussing chamber experiments with T-84 cells and rat jejunum confirm that SP-333 mediated stimulation of short circuit current is dependent on the function of the PKG-II channel and CFTR. In vivo animal model studies further demonstrate that orally administered SP-333 is able to attenuate gastrointestinal transit delay in rats induced by single or repeated opioid doses, as well as attenuate constipation induced by repeated methadone administration.

Mechanism by which GC-C agonists attenuate opioid-induced latency

- 33 040600 gastrointestinal transit and constipation, is shown in FIG. 12. Activation of peripherally expressed opioid receptors by morphine and methadone initiates downstream signals resulting in inhibition of the synthetic cAMP enzyme, adenyl cyclase, and the release of neurotransmitters such as acetylcholine, substance P, and vasoactive gastric peptide (Sobczak et al., 2013). As a result, gastric emptying is delayed, gastro-intestinal transit and peristalsis are weakened, secretion is reduced with a concomitant increase in the absorption of fluids and electrolytes from the intestinal lumen, leading to the manifestation of OIBD (Holzer, 2009; Muller-Lissner, 2010; Sobczak et al., 2013) . Involvement of the apically expressed GC-C receptor of gastric epithelial cells SP-333 promotes the synthesis and accumulation of intracellular cGMP (Zhang et al., 2012). This second communication medium has the potential to influence numerous effectors in cells; they can bind and activate PKG-II, activate protein kinase A (PKA) either directly or indirectly through inhibition of phosphodiesterase 3, which hydrolyzes cAMP to AMP. PKG-II and PKA can phosphorylate and activate the CFTR channel, promoting an efflux of ^CI followed by a passive efflux of Na+ and water into the intestinal lumen, thus facilitating defecation. Electrophysiological data confirm that SP-333-mediated increase in short-circuit current in T-84 cell monolayer and rat jejunal tissues is mediated by PKG-II and CFTR. The presence of a PKA inhibitor (mPKI) and a CIC-2 inhibitor (CdCl ₂ ) has minimal effect on SP-333 stimulated short circuit current in cells and tissues.

Those skilled in the art will understand, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are covered by the claims provided herein.

Inclusion by reference

This application incorporates by reference all publications and references disclosed herein for all purposes in their entirety.

Links.

Barrett KE and Keely SJ (2000) Chloride secretion by the intestinal epithelium: molecular basis and regulatory aspects. Annu Rev Physiol 62:535-572.

Basu N, Arshad N and Visweswariah SS (2010) Receptor guanylyl cyclase C (GC-C): regulation and signal transduction. Mol Cell Biochem 334:67-80.

Bell TJ, Panchai SJ, Miaskowski C, Bolge SC, Milanova T and

Williamson R (2009) The prevalence, severity, and impact of opioid-induced bowel dysfunction: results of a US and European

- 34 040600

Patient Survey (PROBE 1). Pain medicine 10:35-42.

Bianchi G, Fiocchi R, Peracchia F, Petrillo P, Tavani A and Manara L (1984) The peripheral narcotic antagonist N-allyl levallorphan-bromide (CM 32191) selectively prevents morphine antipropulsive action and buprenorphine in-vivo binding in the rat intestine. JPharm Pharmacol 36:326-330.

Bijvelds MJC, Bot AGM, Escher JC and de Jonge HR (2009) Activation of Intestinal CI- Secretion by Lubiprostone Requires the Cystic Fibrosis Transmembrane Conductance Regulator. Gastroenterology 137:976-985.

Camilleri M (201 1) Opioid-induced constipation: challenges and therapeutic opportunities. Am J Gastroenterol 106:835-842; quiz 843.

Clarke LL (2009) A guide to Ussing chamber studies of mouse intestine. Am J Physiol Gastrointest Liver Physiol 296:G1 151-1 166.

Cook SF, Lanza L, Zhou X, Sweeney CT, Goss D, Hollis K, Mangel AW and Fehnel SE (2008) Gastrointestinal side effects in chronic opioid users: results from a population-based survey. Aliment Pharmacol Ther 27: 1224-1232. er BL, Katz S, Vallejo R, Scott CB, Joswick TR, Dolecek G and Ueno R (2010) 906 A Phase 3, Randomized, Double-Blind, Placebo-Controlled Clinical Trial of Lubiprostone for the Treatment of Opioid-Induced Bowel Dysfunction in Patients With Chronic, Non-Cancer Pain. Gastroenterology 138 :S-129.

Cuppoletti J, Chakrabarti J, Tewari K and Malinowska DH (20 13) Methadone but not morphine inhibits lubiprostone-stimulated CI-currents in T84 intestinal cells and recombinant human C1C2, but not CFTR CI-currents. Cell Biochem Biophys 66:53-63.

Cuppoletti J, Chakrabarti J, Tewari KP and Malinowska DH (20 14) Differentiation between Human C1C-2 and CFTR CIChannels with Pharmacological Agents. Am J Physiol Cell Physiol.

Cuppoletti J, Malinowska DH, Tewari KP, Li QJ, Sherry AM, Patchen ML and Ueno R (2004) SPI-02 11 activates T84 cell chloride transport and recombinant human C1C-2 chloride currents. AmJ Physiol Cell Physiol 287 :C1173-1183.

- 35 040600

Fiocchi R, Bianchi G, Petrillo P, Tavani A and Manara L (1982) Morphine inhibits gastrointestinal transit in the rat primarily by impairing propulsive activity of the small intestine. Life Sci 31:222 1-2223.

Forte LR, Jr. (2004) Uroguanylin and guanylin peptides: pharmacology and experimental therapeutics. Pharmacol Ther 104:137-162.

Furlan AD, Sandoval JA, Mailis-Gagnon A and Tunks E (2006) Opioids for chronic noncancer pain: a meta-analysis of effectiveness and side effects. CMAJ 1A 1589-1594.

Golin-Bisello F, Bradbury N and Ameen N (2005) STa and cGMP stimulate CFTR translocation to the surface of villus enterocytes in rat jejunum and is regulated by protein kinase G. Am J Physiol Cell Physiol 289 :C708-7 16.

Holzer P (2009) Opioid receptors in the gastrointestinal tract. Regul Pept 155: 11-17.

Joshi A, Patwa V, Thadi A, Vemalapally L, Foss J, Feng R, Comiskey S, Ricci A, Palejwala V, Brancale A and Shailubhai K (20 14) Plecanatide and SP-333, Novel Homologs of Uroguanylin, Activate Guanylate Cyclase C to Promote Gastrointestinal Motility and Bowel Movement in Rats and Monkeys. Dig Dis Sc z: Communicated.

Ketwaroo GA, Cheng V and Lembo A (20 13) Opioid-induced bowel dysfunction. Curr Gastroenterol Rep 15:344.

Kumar L, Barker C and Emmanuel A (20 14) Opioid-induced constipation: pathophysiology, clinical consequences, and management. Gastroenterology research and practice 2014 :14 1737.

Kurz A and Sessler DI (2003) Opioid-induced bowel dysfunction: pathophysiology and potential new therapies. Drugs 63:649-67 1.

Lipecka J, Bali M, Thomas A, Fanen P, Edelman A and Fritsch J (2002) Distribution of C1C-2 chloride channel in rat and human epithelial tissues. Am J Physiol Cell Physiol 282:0805-8 16.

Love BL, Johnson A and Smith LS (20 14) Linaclotide: A novel agent for chronic constipation and irritable bowel syndrome. Am J Health Syst Pharm 71:1081-109 1.

- 36 040600

Manara L, Bianchi G, Ferretti P and Tavani A (1986) Inhibition of gastrointestinal transit by morphine in rats results primarily from direct drug action on gut opioid sites. J Pharmacol Exp Ther 237:945-949.

Manara L, Bianchi G, Fiocchi R, Notarnicola A, Peracchia F and Tavani A (1982) Inhibition of gastrointestinal transit by morphine and FK 33-824 in the rat and comparative narcotic antagonist properties of naloxone and its N-methyl quaternary analog. Life Sci 31:127 1-1274.

Miner PB, Surowitz R, Fogel R, Koltun W, Drossman DA, Camilleri M, Mangel A, Barrow L, Jacob G and Shailubhai K (2013) Plecanatide, a novel guanylate cyclase-C (GC-C) receptor agonist, is efficacious and safe in patients with chronic idiopathic constipation (CIG): results from a 951 patient, 12 week, multi-center trial. Gastroenterology 144 :S-263 (Abstract 925g).

Miiller-Lissner S (20 10) Opioid-induced Constipation Mechanisms, Relevance and Management. Eur Gastroenterol Hepatol Rev 6:54-57.

Nighot PK, Moeser AJ, Ryan KA, Ghashghaei T and Blikslager AT (2009) C1C-2 is required for rapid restoration of epithelial tight junctions in ischemic-injured murine jejunum. Exp Cell Res 315 : 1 10-1 18.

Pappagallo M (2001) Incidence, prevalence, and management of opioid bowel dysfunction. ArnJ Surg 182: 1 1S-18S.

Pena-Munzenmayer G, Catalan M, Cornejo I, Figueroa CD, Melvin JE, Niemeyer MI, Cid LP and Sepulveda FV (2005) Basolateral localization of native C1C-2 chloride channels in absorptive intestinal epithelial cells and basolateral sorting encoded by a CBS- 2 domain di-leucine motif. J Cell Sci 118:4243-4252.

Penning-van Beest FJ, van den Haak P, Klok RM, Prevoo YF, van der Peet DL and Herings RM (2010) Quality of life in relation to constipation among opioid users. Journal of medical economics 13:129-135.

Quigley C (2005) The role of opioids in cancer pain. BMJ

- 37 040600

331:825-829.

Rauck RL (2013) Treatment of Opioid-Induced Constipation: Focus on the Peripheral mu-Opioid Receptor Antagonist Methylnaltrexone. Drugs 73: 1297-1306.

Shailubhai K (2002) Therapeutic applications of guanylate cyclase-C receptor agonists. Curr Opin Drug Disc Devel 5:261268 .

Shailubhai K, Comiskey S, Foss JA, Feng R, Barrow L, Comer GM and Jacob GS (2013) Plecanatide, an oral guanylate cyclase C agonist acting locally in the gastrointestinal tract, is safe and welltolerated in single doses. DigDis Sci 58:2580-2586.

Shailubhai K, Yu HH, Karunanandaa K, Wang JY, Eber SL, Wang Y, Joo NS, Kim HD, Miedema BW, Abbas SZ, Boddupalli SS, Currie MG and Forte LR (2000) Uroguanylin treatment suppresses polyp formation in the Apc( Min/+) mouse and induces apoptosis in human colon adenocarcinoma cells via cyclic GMP. Cancer Res 60:51515157.

Sobczak M, Salaga M, Storr MA and Fichna J (2013) Physiology, signaling, and pharmacology of opioid receptors and their ligands in the gastrointestinal tract: current concepts and future perspectives. J Gastroenterol.

Steinbrecher KA (2014) The multiple roles of guanylate cyclase C, a heat stable enterotoxin receptor. Current opinion in gastroenterology 30: 1-6.

Steinbrecher KA and Cohen MB (201 1) Transmembrane guanylate cyclase in intestinal pathophysiology. Current opinion in gastroenterology 27:139-145.

Sun X, Wang X, Wang GD, Xia Y, Liu S, Qu M, Needleman BJ, Mikami DJ, Melvin WS, Bohn LM, Ueno R and Wood JD (201 1) Lubiprostone reverses the inhibitory action of morphine on mucosal secretion in human small intestine. Dig Dis Sci 56:330338.

Sykes NP (1998) The relationship between opioid use and laxative use in terminally ill cancer patients. Palliat Med 12:375-382.

Thiagarajah JR, Broadbent T, Hsieh E and Verkman AS (2004)

-

Claims (15)

Prevention of toxin-induced intestinal ion and fluid secretion by a small-molecule CFTR inhibitor. Gastroenterology 126:5 11519. Wang G-D, Wang X, Qu M-H, Xia Y and Wood JD (2014) 414 Reversal of Opioid-Induced Constipation by Lubiprostone (Amitiza®) in Guinea Pig Ileum. Gastroenterology 146:3-89. Wong BS and Camilleri M (201 1) Lubiprostone for the treatment of opioid-induced bowel dysfunction. Expert Opin Pharmacother 12:983-990. Zhang G, Arjunan KP, Foss J, Comiskey S and Shailubhai K (2012) SP-333, a Proteolysis-resistant Agonist of Guanylate Cyclase-C, Inhibits Activation of NF-kappa B and Suppresses Production of Inflammatory Cytokines to Ameliorate DSS-induced Colitis in Mice. Am J Gastroenterol 107 :S626-S626. CLAIM 1. A method of preventing, treating, or alleviating symptoms of opioid-induced bowel dysfunction, comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising a guanylate cyclase receptor agonist (GCRA) peptide consisting of SEQ ID NO: 9, wherein the subject is undergoing chronic opioid use or use of opioids in the acute period. 2. The method of claim 1, further comprising administering an effective dose of a cGMP-dependent phosphodiesterase inhibitor to said subject. 3. The method of claim 2 wherein said cGMP-dependent phosphodiesterase inhibitor is selected from the group consisting of sulindac sulfone, zaprinast, motapizone, vardenafil and sildenafil. 4. The method of claim 2, wherein said cGMP-dependent phosphodiesterase inhibitor is administered either simultaneously or sequentially with said GCRA peptide or pharmaceutical composition thereof. 5. Use of a composition comprising, in a therapeutically effective amount, a guanylate cyclase receptor peptidagonist (GCRA) essentially consisting of SEQ ID NO: 9, for the prevention, treatment, or amelioration of symptoms of opioid-induced bowel dysfunction in a subject in need thereof, wherein the subject is undergoing chronic use opioids or the use of opioids in the acute period. 6. Use according to claim 5, wherein the composition further comprises an effective dose of a cGMP-dependent phosphodiesterase inhibitor. 7. The use of a composition comprising, in a therapeutically effective amount, an opioid and a guanylate cyclase receptor agonist (GCRA) peptide essentially consisting of SEQ ID NO: 9, for the prevention, treatment, or amelioration of symptoms of opioid-induced bowel dysfunction in a subject in need thereof, wherein the subject is undergoing chronic opioid use or acute opioid use. 8. Use according to claim 7, wherein said opioid and guanylate cyclase receptor agonist (GCRA) peptide are administered in a single tablet or capsule. 9. Use according to any one of claims 5 to 7, wherein the composition further comprises one or more guide materials selected from the group consisting of a pH dependent polymer, a swellable polymer, and a degradable composition. 10. Use according to any one of claims 5 to 7, wherein the composition further comprises a pharmaceutical carrier, excipient or diluent. 11. Use according to claim 9, wherein said composition is optimized for delivery to the duodenum, jejunum, ileum, terminal ileum, or ascending colon. 12. Use according to claim 9, wherein said composition is optimized for release throughout the colon. 13. Use according to claim 9, wherein said composition contains one or more pH dependent polymers that degrade in the pH range of 4.5 to 5.5, in the pH range of 5.5 to 6.5, or in the pH range of 6.5 to 7.5. 14. Use according to claim 9, wherein the release of said composition is time dependent. 15. Use according to claim 7, wherein said opioid is selected from the group consisting of morphine, codeine, oxycodone, hydrocodone, dihydrocodeine, propoxyphene, fentanyl, tramadol, loperamide, bu- -