Guanylyl Cyclase C Research Articles

Plecanatide and dolcanatide, novel guanylate cyclase-C agonists, ameliorate gastrointestinal inflammation in experimental models of murine colitis.

To evaluate the effect of orally administered plecanatide or dolcanatide, analogs of uroguanylin, on amelioration of colitis in murine models. The cyclic guanosine monophosphate (cGMP) stimulatory potency of plecanatide and dolcanatide was measured using a human colon carcinoma T84 cell-based assay. For animal studies all test agents were formulated in phosphate buffered saline. Sulfasalazine or 5-amino salicylic acid (5-ASA) served as positive controls. Effect of oral treatment with test agents on amelioration of acute colitis induced either by dextran sulfate sodium (DSS) in drinking water or by rectal instillation of trinitrobenzene sulfonic (TNBS) acid, was examined in BALB/c and/or BDF1 mice. Additionally, the effect of orally administered plecanatide on the spontaneous colitis in T-cell receptor alpha knockout (TCRα(-/-)) mice was also examined. Amelioration of colitis was assessed by monitoring severity of colitis, disease activity index and by histopathology. Frozen colon tissues were used to measure myeloperoxidase activity. Plecanatide and dolcanatide are structurally related analogs of uroguanylin, which is an endogenous ligand of guanylate cyclase-C (GC-C). As expected from the agonists of GC-C, both plecanatide and dolcanatide exhibited potent cGMP-stimulatory activity in T84 cells. Once-daily treatment by oral gavage with either of these analogs (0.05-0.5 mg/kg) ameliorated colitis in both DSS and TNBS-induced models of acute colitis, as assessed by body weight, reduction in colitis severity (P < 0.05) and disease activity index (P < 0.05). Amelioration of colitis by either of the drug candidates was comparable to that achieved by orally administered sulfasalazine or 5-ASA. Plecanatide also effectively ameliorated colitis in TCRα(-/-) mice, a model of spontaneous colitis. As dolcanatide exhibited higher resistance to proteolysis in simulated gastric and intestinal juices, it was selected for further studies. This is the first-ever study reporting the therapeutic utility of GC-C agonists as a new class of orally delivered and mucosally active drug candidates for the treatment of inflammatory bowel diseases.

New Generation Laxatives

A significant proportion of chronic constipation patients are dissatisfied with their treatment. Recently, a number of new medicationshave been introduced for patients refractory to conventional laxatives, such as prucalopride, lubiprostone, linaclotide, andelobixibat. Prucalopride is a novel gastrointestinal prokinetic agent that acts as a 5-hydroxytryptamine type 4 (5-HT4) agonist. Compared with older nonselective 5-HT4 agonists, the higher selectivity of prucalopride for 5-HT4 receptors can reduce the risk ofsignificant adverse cardiovascular events. Prucalopride improves stool frequency and consistency, and reduces the need for rescuemedications. Lubiprostone, a chloride channel activator, increases the secretion of intestinal fluid, improves the stool frequency andconsistency, and reduces straining. Linaclotide, a guanylate cyclase-C agonist, is effective in treating patients with chronic constipationand its effect on visceral sensitivity, as shown mainly in animal studies, provides an attractive pharmaceutical option for patients with irritable bowel syndrome with constipation. Elobixibat is an ileal sodium-dependent bile acid transporter inhibitor thatblocks the enterohepatic circulation of bile acids, increasing the bile acid concentration in the intestine, which accelerates colonictransit and softens the stool. A phase III trial of the treatment of chronic constipation and irritable bowel syndrome with constipationis underway. The clinical application of new-generation laxatives will contribute to the management of chronic constipation refractoryto conventional laxatives.

Prognostic Value of Molecular Detection of Lymph Node Metastases After Curative Resection of Stage II Colon Cancer: A Systematic Pooled Data Analysis

BackgroundWe aimed to clarify the prognostic value of guanylyl cyclase C (GCC) lymph node ratio (LNR) status as a predictor of recurrence in untreated stage IIA colon cancer on the basis of pooled individual data from previous studies. MethodsPatients were classified according to predefined GCC LNR risk groups (low, LNR ≤ 0.1; intermediate, 0.1 < LNR ≤ 0.2; high, LNR > 0.2). Outcomes included time to recurrence, disease-free survival, and overall survival. Stratified log-rank tests and multivariate Cox models assessed the association between outcomes and GCC lymph node status. ResultsThe final data set contained 553 patients with stage IIA colon cancer with a median of 18 lymph nodes examined after resection; 65 patients (11.8%) had recurrence. Overall, 109 patients (19.7%) were classified high risk on the basis of GCC LNR. In multivariate analysis, high GCC LNR value (> 0.2) was a significant predictor of cancer recurrence (hazard ratio [HR], 3.18; 95% confidence interval [CI], 1.77-5.71; P < .001) and lower disease-free survival (HR, 2.40; 95% CI, 1.60-3.62; P < .001) and overall survival (HR, 2.12; 95% CI, 1.35-3.33; P = .001). ConclusionPatients considered at high risk on the basis of their GCC LNR status have significantly inferior outcomes compared to those with low GCC LNR values, particularly among those traditionally considered to be at low risk for recurrence.

Effect of guanylate cyclase-C activity on energy and glucose homeostasis.

Uroguanylin is a gastrointestinal hormone primarily involved in fluid and electrolyte handling. It has recently been reported that prouroguanylin, secreted postprandially, is converted to uroguanylin in the brain and activates the receptor guanylate cyclase-C (GC-C) to reduce food intake and prevent obesity. We tested central nervous system administration of two GC-C agonists and found no significant reduction of food intake. We also carefully phenotyped mice lacking the GC-C receptor and found them to have normal body weight, adiposity, and glucose tolerance. Interestingly, uroguanylin knockout mice had a small but significant increase in body weight and adiposity that was accompanied by glucose intolerance. Our data indicate that the modest effects of uroguanylin on energy and glucose homeostasis are not mediated by central GC-C receptors.

Guanylate Cyclase-C Receptor and Ligand Expression in Colonic Mucosa in Chronic Constipation

Introduction: Linaclotide is a synthetic oral peptide that binds guanylate cyclase C (GC-C) receptors in the intestinal epithelium and has demonstrated efficacy in chronic idiopathic constipation (CC). Guanylin and uroguanylin stimulate GC-C receptors which increases intracellular levels of cGMP resulting in intestinal fluid secretion and acceleration of transit time. Cyclic GMP (cGMP) transporter proteins function as an ATP-driven export pump for cyclic AMP and cGMP. The expression of GC-C receptor and ligand, uroguanylin, and guanylin, and the cGMP transporters in the human colon has not been well studied. Aims: 1) To compare the mRNA expression and protein levels of the GC-C receptor, guanylin and uroguanylin, cGMP transporter proteins, multidrug resistance protein 4 and 5 (MRP4 and MRP5) in the colonic mucosa of female patients with CC and healthy controls and, 2) To determine if there is an association with overall GI symptoms. Methods: Women between the ages of 18-55 were recruited mainly from advertisement with: 1) Rome III positive CC (30.7±11.8 years), and 2) healthy controls (34.6±10.1 years) (n=12 in each group). Subjects completed GI symptom questionnaires including overall GI symptom severity using a 21-point numeric rating scale (0-20). All subjects underwent a flexible sigmoidoscopy with random biopsies taken at 30 cm from the anal verge. RT-PCR, ELISA assays and Western blots were performed to measure mRNA expression and protein levels, respectively. Student t-tests or non-parametric Wilcoxon rank sum tests were used for group comparisons. Spearman correlation was also used. A p value of <0.05 was considered significant. Results: There were no significant group differences in mRNA expression of the GC-C receptor, GCC receptor agonists (guanylin and uroguanylin) and the cGMP transporter proteins. Similar findings were found with respect to protein levels, except for MRP4, which was significantly higher in CC patients (0.043 pg/μg) vs. controls (0.034 pg/μg) (p=0.01). The CC patients had a moderate GI symptom severity (mean 10.45±5.11 [0-20]). There was a significant negative correlation between guanylin protein levels and current overall GI symptom severity in CC patients (r= -0.701, p=0.024). Conclusion: Findings suggest that the GC-C signaling pathway plays a role in the pathogenesis and symptomatology in CC. Further studies in CC are needed to determine: 1) if mucosal guanylin levels are predictive of severity or treatment response to a GC-C agonist, and 2) the significance of increased expression of MRP4, e.g., if mucosal levels are predictive of the response to a constipation medication given that MRP4 is a drug transporter protein and confers resistance to a variety of drugs. Disclosure - Lin Chang- Consultant and Grant Support: Ironwood, Consultant: Forest. This research was supported by an industry grant from Ironwood Pharmaceuticals

Effect of Diarrhea Adverse Events on Health-Related Quality of Life and Treatment Satisfaction in Patients With Chronic Idiopathic Constipation

Introduction: To evaluate the effect of diarrhea adverse events (AEs) on health-related quality of life (HRQOL) and treatment satisfaction (TS) in patients with chronic idiopathic constipation (CIC). Methods: Data were pooled from 2 similarly designed phase 3 trials in CIC patients of linaclotide, the first-in-class guanylate cyclase-C agonist FDA-approved treatment for adults with CIC and irritable bowel syndrome with constipation. Presence and severity (ie, mild, moderate, severe) of diarrhea episodes were recorded by trial investigators as AEs at any time during the 12-week treatment period in both trials. HRQOL was assessed at baseline and week 12 using the patient assessment of constipation quality of life questionnaire (PAC-QOL) overall score (range: 0-4, lower scores indicate better HRQOL); TS was evaluated at weeks 2, 4, 8, and 12 with a 5-point scale (1=not at all satisfied, 5=very satisfied). Data were evaluated longitudinally by pairing the most severe diarrhea episode with the nearest HRQOL/TS assessment following the episode. Change-from-baseline to week 12 in PAC-QOL overall score and mean TS score at week 12 were evaluated by treatment (linaclotide 145 mcg vs. placebo) stratified by diarrhea presence/severity. Patients with no recorded diarrhea episodes were the reference group. Longitudinal multivariate models examined adjusted associations between diarrhea episodes and HRQOL/TS, controlling for patient characteristics and change in abdominal (pain, bloating, discomfort) and bowel symptoms (stool frequency, stool consistency, straining). Results: Of 1,276 patients randomized, 854 received a treatment of interest for these analyses (linaclotide 145 mcg n=430, placebo n=424), of which 746 completed HRQOL and TS assessments (linaclotide 145 mcg n=369, placebo n=377). Of these, 105 diarrhea episodes were reported in 98 patients (n=51 mild, n=46 moderate, n=8 severe). Mean TS and change in PAC-QOL overall scores did not significantly differ in linaclotide or placebo patients with diarrhea AEs vs. those with no recorded diarrhea AEs (TS=3.52 vs. 3.28; p=0.269 for linaclotide and 2.80 vs. 2.21; p=0.185 for placebo; change in PAC-QOL=-0.96 vs. -0.95; p=0.340 for linaclotide and -0.98 vs. -0.57; p=0.207 for placebo). For both linaclotide and placebo, the presence of diarrhea (any severity) had no statistically significant impact on TS or HRQOL, even after controlling for confounders. Conclusion: Presence of diarrhea AEs did not negatively affect HRQOL or TS among CIC patients receiving linaclotide 145 mcg. Disclosure - Michael Munsell and Robert Griffiths are employees of Boston Health Economics, Inc, which is a consultancy whose activities related to the project are funded by Forest Research Institute, and Ironwood Pharmaceuticals; Huan Huang is a consultant to Ironwood Pharmaceuticals, Inc., and Forest Research Institute; Jessica Buono and Robyn Carson are employees of Forest Research Institute and own stock and/or stock options in Forest Laboratories, Inc.; William Spalding and Douglas Taylor are employees of Ironwood Pharmaceuticals, Inc., and own stock and/or stock options in Ironwood Pharmaceuticals. This research was supported by an industry grant from Forest Research Institute and Ironwood Pharmaceuticals.

SP-333, a D-Amino Acid Containing Peptide Agonist of Guanylate Cyclase-C Is a Novel Drug Candidate for Treatment of Gastrointestinal Disorders and Diseases

SP-333, a D-Amino Acid Containing Peptide Agonist of Guanylate Cyclase-C Is a Novel Drug Candidate for Treatment of Gastrointestinal Disorders and Diseases

Effect of Diarrhea Adverse Events on Health-Related Quality of Life and Treatment Satisfaction in Patients With Irritable Bowel Syndrome With Constipation

Introduction: To evaluate the effect of diarrhea adverse events (AEs) on health-related quality of life (HRQOL) and treatment satisfaction (TS) in patients with irritable bowel syndrome with constipation (IBS-C). Methods: Data were pooled from 2 similarly designed phase 3 trials in IBS-C patients of linaclotide, the first-in-class guanylate cyclase-C agonist FDA-approved treatment for adults with IBS-C or chronic idiopathic constipation. Presence and severity (i.e., mild, moderate, severe) of diarrhea episodes were recorded by trial investigators as AEs at any time during the 12-week treatment period in both trials. HRQOL was assessed at baseline and week 12 using the irritable bowel syndrome quality of life (IBSQOL) questionnaire overall score (range: 0-100, higher scores indicate better HRQOL); TS was evaluated at weeks 2, 4, 8, and 12 with a 5-point scale (1=not at all satisfied, 5=very satisfied). Data were evaluated longitudinally by pairing the most severe diarrhea episode with the nearest HRQOL/TS assessment following the episode. Change-from-baseline to week 12 in IBS-QOL overall score and mean TS at week 12 were evaluated by treatment (linaclotide 290 mcg vs. placebo) stratified by diarrhea presence/severity. Patients with no recorded diarrhea episodes were the reference group. Longitudinal multivariate models examined adjusted associations between diarrhea episodes and HRQOL/TS, controlling for patient characteristics and change in abdominal (e.g., pain, bloating, discomfort) and bowel symptoms (stool frequency, stool consistency, straining). Results: Of 1,602 patients randomized, 1,336 completed HRQOL and TS assessments (linaclotide 290 mcg n=654, placebo n=682); of these, 129 diarrhea episodes were reported in 122 patients (n=69 mild, n=53 moderate, n=7 severe). Mean TS and change in IBS-QOL did not significantly differ in linaclotide patients with diarrhea AEs vs. those with no recorded diarrhea AEs (TS=3.54 vs. 3.40; p=0.433; change in IBS-QOL=18.47 vs. 19.06; p=0.408, respectively). Change in IBS-QOL also did not significantly differ for placebo patients with diarrhea AEs vs those with no recorded diarrhea AEs (8.54 vs. 13.58; p=0.931, respectively). For both linaclotide and placebo, the presence of diarrhea (any severity) had no statistically significant impact on HRQOL, even after controlling for confounders. A statistically significant impact on TS was found among linaclotide patients with a recorded episode of severe diarrhea (-0.80, p=0.001); among placebo patients, mild (-0.40, p=0.042) and severe (-1.55, p=0.046) diarrhea AEs had a significant impact. Conclusion: Presence of mild/moderate diarrhea AEs did not negatively affect HRQOL or TS among IBS-C patients receiving linaclotide 290 mcg. Disclosure - Michael Munsell and Robert Griffiths are employees of Boston Health Economics, Inc, which is a consultancy whose activities related to the project are funded by Forest Research Institute, and Ironwood Pharmaceuticals; Huan Huang is a consultant to Ironwood Pharmaceuticals, Inc., and Forest Research Institute; Jessica Buono and Robyn Carson are employees of Forest Research Institute and own stock and/or stock options in Forest Laboratories, Inc.; William Spalding and Douglas Taylor are employees of Ironwood Pharmaceuticals, Inc., and own stock and/or stock options in Ironwood Pharmaceuticals. This research was supported by an industry grant from Forest Research Institute and Ironwood Pharmaceuticals.

Plecanatide and SP-333, Novel Agonists of Guanylate Cyclase-C, Attenuate Visceral Hypersensitivity in Rat Models

Plecanatide and SP-333, Novel Agonists of Guanylate Cyclase-C, Attenuate Visceral Hypersensitivity in Rat Models

Abstract 4949: In vitro and in vivo investigation of the novel, first-in-class, Guanylyl Cyclase C (GCC) targeted 68Ga labeled heat stable peptide MLN6907 ([68Ga]MLN6907) for tumor imaging

Abstract A novel, first-in-class, 68Ga labeled DOTA-para-bn-SCN-Ahx-STp(5-18) 2.2kDa peptide [MLN6907] ([68Ga]MLN6907) with high affinity to the guanalyl cyclase C (GCC) receptor has been developed to select cancer patients for treatment with GCC targeted therapies using PET/CT. GCC is sequestered exclusively in the gastrointestinal luminal compartment except under malignant transformation where it is then made accessible to intravenous agents. Conventional patient selection strategies often rely on an analytical IHC or total protein based assessment of a tumor biopsy which can be limited to an archival tissue sample from a single region of a single lesion. Imaging may offer whole body, real time, multi-region and multi lesion assessment of target levels. In addition, functional parameters associated with receptor kinetics may also be explored in vivo. 68Ga has a high avidity for the common chelating moiety DOTA. Furthermore, its short radioactive half life (half-life, 68.3 minutes) matches well to a biological targeting moiety like a peptide with its rapid biological clearance and fast diffusion to target thus providing optimal tumour-to-normal tissue contrast. In vitro: Cellular competitive binding studies confirmed high affinity of non-radiolabeled MLN6907 for its cognate receptor, GCC, with a KD of 3.2 nM. Upon ligand-recpetor binding, MLN6907 is internalized rapidly with a half-life of 56 min. In vivo: Similar to other radiolabeled peptides, [68Ga]MLN6907 clears rapidly from blood (t1/2 = 26 min) through renal excretion as investigated in Long Evans rats and non-human primate studies. The radiation exposure from [68Ga]MLN6907 was highest in the kidney and bladder indicating that renal excretion was a primary route of elimination. Using OLINDA/EXM software, the effective dose was estimated to be 0.013 mSv/MBq in man. In tumor bearing C.B-17 SCID mice, both ex vivo and in vivo signal was measured in both human tumor cell lines and primary human tumor xenografts with varied GCC levels and compared to non tumor bearing tissues. [68Ga]MLN6907 total uptake (%I.D./g) varied across the different tumors investigated. Using the unlabeled precursor, no toxicity was observed in repeat dose rat and monkey studies. Using the rat as the more conservative species for dose estimation, it was calculated that a human equivalent dose of 282 μg would be safe. However, using a saturable effect PK/PD compartment model for mice and humans, we estimated a single human dose of less than 100μg would provide resolution of tumors with different GCC levels. Collectively, biological, pharmacokinetic and safety data obtained with [68Ga]MLN6907 are consistent with an effective GCC PET imaging agent. [68Ga]MLN6907 is being developed clinically as a single, i.v., microdose GCC PET imaging agent in a Phase 1 investigation in patients with surgically resectable metastatic colorectal carcinoma Citation Format: Donna Cvet, Robert Robertson, Melissa Saylor, Jennifer Terkelsen, Ozlem Yardibi, Maria Borland, Nicolas Salem, Petter Veiby, Todd Sells, Mary Carsillo, Johnny Yang, Shu-Wen Teng, John Hoppin, Kelly Orcutt, Jacob Hesterman, Jeffery Norenberg, Tamara Anderson, Mike Schulz, Mary Ruscowski, Marc Berridge, Steven Mather, Daniel P. Bradley. In vitro and in vivo investigation of the novel, first-in-class, Guanylyl Cyclase C (GCC) targeted 68Ga labeled heat stable peptide MLN6907 ([68Ga]MLN6907) for tumor imaging. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4949. doi:10.1158/1538-7445.AM2014-4949

Abstract 4649: Using pharmacokinetic/efficacy modeling to identify the optimal schedule for MLN0264, an anti- guanylyl cyclase C (GCC) antibody-drug conjugate, in a range of xenograft models

Abstract MLN0264 is an investigational antibody-drug conjugate (ADC) that consists of the human anti-guanylyl cyclase C (GCC) antibody linked to a microtubule-disrupting agent (monomethyl auristatin). As ADCs have a very long clearance half-life, the potential exists for a highly infrequent dosing schedule. A quantitative understanding of the relationship between exposure and preclinical antitumor biological activity is thus applied to support dose schedule selection in the clinic. In this study, we develop a pharmacokinetic/efficacy (PK/E) relationship in xenograft models to evaluate the predictive contributions of exposure and xenograft characteristics to MLN0264 biological activity. Single dose pharmacokinetic (PK) data were obtained for a range of time points, and a linear two-compartment PK model was built. Xenograft biological activity studies were conducted in which MLN0264 was administered at various dose levels and dosing schedules to mice bearing one of six different xenograft models. We used multiple linear regression and tumor dynamic modeling to understand the factors contributing to the biological activity. First, the growth rate of each tumor under control and treatment conditions was established by fitting the biological activity data within the dosing period to an exponential growth function. Then, we assessed the relationship of AUC to antitumor biological activity. Within each xenograft model, AUC was strongly correlated with biological activity across a range of schedules (R2&amp;gt;0.9, p&amp;lt;0.03, for all models). AUC-proportional biological activity was confirmed using dynamic modeling with direct drug effect to simulate the tumor dynamics for different dosing schedules. Although biological activity within a xenograft model was strongly correlated with exposure, when the data from different xenograft models were pooled together, the correlation was weaker (R2= 0.30, p= 0.0004). This result suggests that, although schedule is not a major determinant of biological activity, other xenograft-specific factors may be contributing to biological activity. To test the contribution of some possible factors, we used multiple linear regression to determine which covariates (such as GCC expression level and baseline growth rate of xenograft models) are predictive of biological activity. The results suggest baseline growth rate does not correlate with biological activity while GCC expression is weakly correlated. This finding formed the basis for the development of a mechanistic model of GCC expression and biological activity. Taken together, this work demonstrates the use of a PK/E framework to identify the scheduling effect for a first-in-man protocol for an ADC. We have also demonstrated that this PK/E framework can be further leveraged to assess the contribution of other potential predictors of ADC biological activity in xenograft models. Citation Format: Shu-Wen Teng, Christopher Zopf, Johnny Yang, Brad Stringer, Julie Zhang, Wen Chyi Shyu, Arijit Chakravarty, Petter Veiby, Jerome Mettetal. Using pharmacokinetic/efficacy modeling to identify the optimal schedule for MLN0264, an anti- guanylyl cyclase C (GCC) antibody-drug conjugate, in a range of xenograft models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4649. doi:10.1158/1538-7445.AM2014-4649

Abstract 4948: PET/CT clinical protocol design for the novel, first in class 68Ga labeled guanylyl cyclase C targeted peptide MLN6907 ([68Ga]MLN6907)

Abstract Acquisition and interpretation guidelines for clinical PET/CT imaging in oncology have been designed for whole-body 18F-FDG imaging and may not be optimized for assessment of other PET imaging tracers. Here we describe a methodology of PET/CT study design for the novel first in class 68Ga-labeled Guanylyl cyclase C (GCC) targeted peptide, [68Ga]MLN6907, based on a combination of in vitro, ex vivo, and in vivo preclinical imaging studies and model-based estimation of tumor parameters from simulated clinical PET data. GCC, a protein expressed in GI malignancies, is being targeted by the antibody drug conjugate MLN0264. GCC is also expressed on the healthy apical surface of the intestinal epithelium, which should be inaccessible to intravascular treatment. [68Ga]MLN6907 binds GCC with high affinity and is being developed as an imaging biomarker in an effort to help identify patients likely to respond to GCC-targeted therapy. In a series of experiments, the peptide affinity, internalization rate, and clearance were determined in patient-derived CRC xenografts with varied tumor microenvironmental phenotype. In addition to supporting the clinical development of the imaging agent, this data was used in combination with simulated clinical list-mode PET data to evaluate tumor parameter estimability under several clinically viable acquisition and reconstruction conditions. Specifically, liver CRC metastases of varying tumor diameter, antigen density, and vascularity were simulated in combination with PET imaging acquisition duration and reconstruction with and without partial-volume correction. Tumor, liver, and background time-activity curves (TACs) were generated from the reconstructed data and analyzed using a distributed tumor model to estimate the known tumor antigen density and vascularity. Analysis of the simulation studies revealed: 1) Partial volume correction is required for accurate antigen density and vascularity estimation; 2) Parameter estimation was most accurate within a tumor size range of 1-5 cm; 3) Parameter estimation was robust for all tested TAC reconstruction durations (e.g., 2, 3, 5, and 10 min); 4) Parameter estimation was optimal for common clinical acquisition times of 30-90 minutes; 5) Antigen density estimation was less accurate in poorly vascularized tumors. For the translation of a novel clinical biomarker, well controlled preclinical studies are critical; and, in this case, the findings combined with the distributed tumor model simulations directly guided the clinical image protocol. This rational and data-driven approach has the ability to not only improve the estimation of tumor properties in human subjects but also to guide the design of first-in-human oncology clinical imaging protocols with novel biomarkers. Citation Format: Jacob Y. Hesterman, Kelly D. Orcutt, Ozlem Yardibi, Jerome T. Mettetal, Shu-Wen Teng, Donna Cvet, Jack Hoppin, Thea Kalebic, Daniel P. Bradley. PET/CT clinical protocol design for the novel, first in class 68Ga labeled guanylyl cyclase C targeted peptide MLN6907 ([68Ga]MLN6907). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4948. doi:10.1158/1538-7445.AM2014-4948

GUCY2C (guanylate cyclase 2C (heat stable enterotoxin receptor))

Review on GUCY2C (guanylate cyclase 2C (heat stable enterotoxin receptor)), with data on DNA, on the protein encoded, and where the gene is implicated.

Molecular testing for lymph node metastases as a determinant of colon cancer recurrence: results from a retrospective multicenter study.

Recurrence risk assessment to make treatment decisions for early-stage colon cancer patients is a major unmet medical need. The aim of this retrospective multicenter study was to evaluate the clinical utility of guanylyl cyclase C (GCC) mRNA levels in lymph nodes on colon cancer recurrence. The proportion of lymph nodes with GCC-positive mRNA (LNR) was evaluated in 463 untreated T3N0 patients, blinded to clinical outcomes. One site's (n = 97) tissue grossing method precluded appropriate lymph node assessment resulting in post hoc exclusion. Cox regression models tested the relationship between GCC and the primary endpoint of time to recurrence. Assay methods, primary analyses, and cut points were all prespecified. Final dataset contained 366 patients, 38 (10%) of whom had recurrence. Presence of four or more GCC-positive lymph nodes was significantly associated with risk of recurrence [hazard ratio (HR) = 2.46, 95% confidence interval (CI), 1.07-5.69, P = 0.035], whereas binary GCC LNR risk class (HR = 1.87, 95% CI, 0.99-3.54, P = 0.054) and mismatch repair (MMR) status (HR = 0.77, 95% CI, 0.36-1.62, P = 0.49) were not. In a secondary analysis using a 3-level GCC LNR risk group classification of high (LNR > 0.20), intermediate (0.10 < LNR ≤ 0.20), and low (LNR ≤ 0.10), high-risk patients had a 2.5 times higher recurrence risk compared with low-risk patients (HR = 2.53, 95% CI, 1.24-5.17, P = 0.011). GCC status is a promising prognostic factor independent of traditional histopathology risk factors in a contemporary population of patients with stage IIa colon cancer not treated with adjuvant therapy, but GCC determination must be performed with methodology adapted to the tissue procurement and fixation technique.

Uroguanylin inhibits H-ATPase activity and surface expression in renal distal tubules by a PKG-dependent pathway

Cumulative evidence suggests that guanylin peptides play an important role on electrolyte homeostasis. We have previously reported that uroguanylin (UGN) inhibits bicarbonate reabsorption in a renal distal tubule. In the present study, we tested the hypothesis that the bicarbonaturic effect of UGN is at least in part attributable to inhibition of H(+)-ATPase-mediated hydrogen secretion in the distal nephron. By in vivo stationary microperfusion experiments, we were able to show that UGN inhibits H(+)-ATPase activity by a PKG-dependent pathway because KT5823 (PKG inhibitor) abolished the UGN effect on distal bicarbonate reabsorption and H89 (PKA inhibitor) was unable to prevent it. The in vivo results were confirmed by the in vitro experiments, where we used fluorescence microscopy to measure intracellular pH (pHi) recovery after an acid pulse with NH4Cl. By this technique, we observed that UGN and 8 bromoguanosine-cGMP (8Br-cGMP) inhibited H(+)-ATPase-dependent pHi recovery and that the UGN inhibitory effect was abolished in the presence of the PKG inhibitor. In addition, by using RT-PCR technique, we verified that Madin-Darby canine kidney (MDCK)-C11 cells express guanylate cyclase-C. Besides, UGN stimulated an increase of both cGMP content and PKG activity but was unable to increase the production of cellular cAMP content and PKA activity. Furthermore, we found that UGN reduced cell surface abundance of H+-ATPase B1 subunit in MDCK-C11 and that this effect was abolished by the PKG inhibitor. Taken together, our data suggest that UGN inhibits H(+)-ATPase activity and surface expression in renal distal cells by a cGMP/PKG-dependent pathway.

Association between GUC2C and ADHD: Evidence from both categorical and quantitative traits

Guanylyl cyclase-C knock-out mice exhibit hyperactivity and attention deficits. We investigated seven single nucleotide polymorphisms (SNPs) of Guanylate cyclase 2C gene (GUC2C) in 381 ADHD trios and 382 healthy controls. Transmission disequilibrium tests (TDT), case-control studies and quantitative analyses indicated association between GUC2C with ADHD and its core symptoms.

Linaclotide: a novel agent for chronic constipation and irritable bowel syndrome.

The pharmacology, pharmaco-kinetics, and clinical efficacy and safety of linaclotide in the management of chronic constipation (CC) and constipation-predominant irritable bowel syndrome (IBS-C) are reviewed. Linaclotide (Linzess, Forest Pharmaceuticals) is a 14-amino acid peptide indicated for the treatment of adults with CC and IBS-C. Linaclotide acts on guanylate cyclase-C receptors on the luminal membrane to increase chloride and bicarbonate secretions into the intestine and inhibit the absorption of sodium ions, thus increasing the secretion of water into the lumen and improving defecation; the drug is minimally absorbed into the systemic circulation. Linaclotide is approved by the Food and Drug Administration (FDA) for oral once-daily administration at doses of 145 μg for CC and 290 μg for IBS-C. In placebo-controlled Phase III clinical trials, linaclotide significantly increased weekly spontaneous bowel movements and complete spontaneous bowel movements (CSBMs) while reducing abdominal pain in patients with CC. In patients with IBS-C, linaclotide was demonstrated to be effective in meeting FDA-recommended endpoints such as reductions of at least 30% from baseline in abdominal pain scores and CSBM frequency. The most common adverse effect of linaclotide is diarrhea, which was reported in 16-20% of clinical trial participants. Linaclotide is an important advance in the treatment of CC and IBS-C, with a novel mechanism of action resulting in accelerated intestinal transit. In clinical trials, linaclotide demonstrated efficacy relative to placebo for treatment of both CC and IBS-C. Linaclotide's adverse effects are generally mild and confined to the gastrointestinal tract.

PWE-167 Effect Of Linaclotide On Ibs-qol Sexual Subscale Scores In Patients With Irritable Bowel Syndrome With Constipation: Results From 2 Phase 3 Trials

IntroductionLinaclotide is a minimally absorbed guanylate cyclase-C agonist approved for treatment of IBS with constipation (IBS-C). IBS often results in diminished quality of life (QOL), including decreased sexual desire and...

Mo2029 Extracellular Cyclic GMP (cGMP), the Downstream Mediator Released in Response to Linaclotide-Induced Activation of Guanylate Cyclase-C (GC-C), Reduces Excitability of Murine and Human Dorsal Root Ganglion (DRG) Neurons

G A A b st ra ct s that was performed every 4 days to determine the exact timepoint of endoscopic healing in each individual animal. Three days after endoscopic resolution of inflammation, visceral sensitivity was assessed by quantifying visceromotor responses (VMRs) to colorectal distension. A-317491 (10-25 mg/kg) or saline was administered 30 min prior to VMR testing. Compliance was evaluated by applying graded volumes to the colorectally inserted balloon and recording the corresponding colonic pressures. Afterwards remaining signs of inflammation were assessed by endoscopy, macroscopy, histology and myeloperoxidase activity. P2X3R mRNA expression was determined in colon specimens and dorsal root ganglia (DRGs; Th13-L2 and L6-S1) by RT-PCR. Results: Endoscopy demonstrated that colitis was present 3 days after the TNBS enema and had resolved by day 12 (range 10-14); inflammatory markers were normal in control and post-colitis rats at the time of VMR testing. After endoscopic healing of colitis, post-colitis rats displayed significant visceral hypersensitivity compared to controls (table 1). Post-inflammatory hypersensitivity was dose-dependently reduced by A-317491 without affecting VMRs in controls. Colonic compliance was comparable in both groups and remained unaltered by A-317491 (n=5-7). P2X3RmRNAwas similarly expressed in the colon and DRGs of control and post-colitis rats. Conclusion: The selective P2X3R antagonist A-317491 dose-dependently reduced post-inflammatory visceral hypersensitivity, highlighting the modulatory role of P2X3Rs in visceral nociception. Our findings validate this receptor subtype as a potential target in the treatment of abdominal pain syndromes such as irritable bowel syndrome and inflammatory bowel disease. Table 1. Visceromotor responses to colorectal distension in control and post-colitis rats.

Sa2013 Oral Treatment With SP-333, an Agonist of Guanylate Cyclase-C, Dramatically Ameliorates Methadone-Induced Bowel Dysfunction in Rats

Sa2013 Oral Treatment With SP-333, an Agonist of Guanylate Cyclase-C, Dramatically Ameliorates Methadone-Induced Bowel Dysfunction in Rats