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Strategies to augment immunity to self/neoantigens expressed by cancers are urgently needed to expand the proportion of patients benefiting from immunotherapy, particularly for GI cancers where only a fraction of patients respond to immunotherapies. However, current vaccine strategies are limited by poor immunogenicity, pre-existing vector-specific immunity, and vaccine-induced vector-specific immunity. Here, we examined a prime-boost strategy using a chimeric adenoviral vector (Ad5.F35) that resists pre-existing immunity followed by recombinant Listeria monocytogenes (Lm) to amplify immunity to the GI cancer antigen GUCY2C. This previously unexplored combination enhanced the quantity, avidity, polyfunctionality, and antitumor efficacy of GUCY2C-specific effector CD8+ T cells, without toxicity in any tissue, including GUCY2C-expressing intestines and brain. Importantly, this combination was partially resistant to pre-existing immunity to Ad5 which is endemic in human populations and vector-specific immunity did not limit the ability of multiple Lm administrations to repeatedly enhance GUCY2C-specific responses. Broadly, these findings suggest that cancer patient immunizations targeting self/neoantigens, as well as immunizations for difficult infectious diseases (HIV, malaria, etc), may be most successful using a combination of Ad5.F35-based priming, followed by Lm-based boosting. More specifically, Lm-GUCY2C may be utilized to amplify GUCY2C-specific immunity in patients receiving adenovirus-based GUCY2C vaccines currently in clinical trials to prevent or treat recurrent GI cancer.

Personalized medicine epitomizes an evolving model of care tailored to the individual patient. This emerging paradigm harnesses radical technological advances to define each patient's molecular characteristics and decipher his or her unique pathophysiological processes. Translated into individualized algorithms, personalized medicine aims to predict, prevent, and cure disease without producing therapeutic adverse events. Although the transformative power of personalized medicine is generally recognized by physicians, patients, and payers, the complexity of translating discoveries into new modalities that transform health care is less appreciated. We often consider the flow of innovation and technology along a continuum of discovery, development, regulation, and application bridging the bench with the bedside. However, this process also can be viewed through a complementary prism, as a necessary supply chain of services and providers, each making essential contributions to the development of the final product to maximize value to consumers. Considering personalized medicine in this context of supply chain management highlights essential points of vulnerability and/or scalability that can ultimately constrain translation of the biological revolution or potentiate it into individualized diagnostics and therapeutics for optimized value creation and delivery.

Guanylyl cyclase C (GUCY2C) is the index cancer mucosa antigen, an emerging class of immunotherapeutic targets for the prevention of recurrent metastases originating in visceral epithelia. GUCY2C is an autoantigen principally expressed by intestinal epithelium, and universally by primary and metastatic colorectal tumors. Immunization with adenovirus expressing the structurally unique GUCY2C extracellular domain (GUCY2C(ECD); Ad5-GUCY2C) produces prophylactic and therapeutic protection against GUCY2C-expressing colon cancer metastases in mice, without collateral autoimmunity. GUCY2C antitumor efficacy is mediated by a unique immunological mechanism involving lineage-specific induction of antigen-targeted CD8(+) T cells, without CD4(+) T cells or B cells. Here, the unusual lineage specificity of this response was explored by integrating high-throughput peptide screening and bioinformatics, revealing the role for GUCY2C-directed CD8(+) T cells targeting specific epitopes in antitumor efficacy. In BALB/c mice vaccinated with Ad5-GUCY2C, CD8(+) T cells recognize the dominant GUCY2C(254-262) epitope in the context of H-2K(d), driving critical effector functions including interferon gamma secretion, cytolysis ex vivo and in vivo, and antitumor efficacy. The ability of GUCY2C to induce lineage-specific responses targeted to cytotoxic CD8(+) T cells recognizing a single epitope mediating antitumor efficacy without autoimmunity highlights the immediate translational potential of cancer mucosa antigen-based vaccines for preventing metastases of mucosa-derived cancers.

For the last decade, we have focused on guanylyl cyclase C (GUCY2C) as a potentially ideal target antigen for colorectal cancer immunotherapy. GUCY2C is expressed only in intestinal epithelial cells and by nearly 100% of colorectal cancers. We have developed and tested a recombinant adenoviral vector possessing GUCY2C (Ad5-GUCY2C) as a candidate vaccine for colorectal cancer patients. Murine studies have revealed that this vaccine is safe and effective against GUCY2C-expressing targets, and Ad5-GUCY2C is poised for phase I clinical testing in colorectal cancer patients with minimal residual disease. Moreover, we are developing second-generation GUCY2C-targeted therapeutics, including the use of chimeric antigen receptor (CAR)-expressing T cells, for treatment of patients with advanced colorectal cancer for whom Ad5-GUCY2C immunization is not appropriate. Thus, a family of GUCY2C-targeted immunotherapeutics may bridge the gap in effective treatments for the 500,000 patients worldwide who die annually from colorectal cancer.

1Department of Pharmacology and Experimental Therapeutics, Division of Clinical Pharmacology, Department of Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania, USA; 2Departments of Internal Medicine and Pharmacology, University of Iowa, Iowa City, Iowa, USA; 3Divisions of Pediatric Pharmacology and Medical Toxicology, Children’s Mercy Hospitals and Clinics, Kansas City, Missouri, USA; 4American Society for Clinical Pharmacology and Therapeutics, Alexandria, Virginia, USA; 5Divisions of Cardiovascular Diseases and Clinical Pharmacology, Departments of Medicine, Molecular Pharmacology and Experimental Therapeutics and Medical Genetics, Mayo Clinic, Rochester, Minnesota, USA. Correspondence: SA Waldman (scott.waldman@jefferson.edu) or A Terzic (terzic.andre@mayo.edu)

Obesity has emerged as one of the principal worldwide health concerns of the modern era, and there exists a tremendous unmet clinical need for safe and effective therapies to combat this global pandemic. The prevalence of obesity and its associated comorbidities, including cardiovascular and metabolic diseases, has focused the attention of those in drug discovery and development on generating effective modalities for the treatment and prevention of obesity. Early efforts in the field of obesity pharmacotherapy centered on the development of agents with indeterminate mechanisms of action. This led to treatment paradigms characterized by significant off-target effects. In the past two decades, new insights have been made into the physiologic regulation of energy balance and the subordinate central and peripheral circuits coordinating appetite, metabolism, and lipogenesis. These studies have revealed previously unrecognized molecular targets for controlling appetite and managing weight from which has emerged a new wave of targeted pharmacotherapies to prevent and control obesity. Clinical Pharmacology & Therapeutics (2010) 87 6, 652–662. doi: 10.1038/clpt.2010.57

Colorectal cancer is a leading cause of cancer-related mortality worldwide. Surgery and chemoradiation exhibit incomplete efficacy and, ultimately, 50% of patients die of metastatic disease. In the context of that unmet clinical need, immunotherapeutic approaches have enjoyed limited success, partly because of a paucity of suitable antigen targets. However, exploitation of immune compartmentalization, employing antigens with expression restricted to normal intestinal mucosa and derivative colorectal tumors--cancer mucosa antigens (CMAs)--may represent a previously unrecognized class of immune targets supporting efficacious antitumor immunotherapy. Guanylyl cyclase C (GCC) is an intestine/colorectal cancer-restricted protein ideally suited as the first CMA for clinical evaluation.

Colorectal carcinogenesis originates in the context of dysregulated epithelial cell homeostasis, wherein hyperproliferation, hypodifferentiation, metabolic reprogramming, and mesenchymal remodeling reflect recursive mutually reinforcing mechanisms contributing to progressive genomic instability. Although genotypic and phenotypic elements characterizing the terminal integration of these pathophysiological processes defining cancer are well enumerated, events initiating, coordinating, and sustaining this hierarchical maladaptive systems evolution remain elusive for most tumors. In the intestine, guanylyl cyclase C (GCC) and its paracrine ligands organize and regulate the homeostatic integrity of the crypt-villus axis, forming a hormonal tumor suppressor signaling sequence, whose dysfunction defines the initiation of neoplastic transformation and creates a permissive niche for tumor progression.

Colorectal cancer is the fourth most common cancer and the second leading cause of cancer death. Transformation begins with activation of Wnt signaling through mutations in APC (80%) or its degradation target β-catenin (15%), producing a gain-of-function in TCF-dependent nuclear transcription underlying epithelial dysfunction and tumorigenesis. While a role for APC and β-catenin in colorectal cancer is well-established, steps leading from gene mutation to tumorigenesis, and their reversibility, remain incompletely defined. Guanylin is the paracrine hormone in colorectum for the receptor GUCY2C. This hormone is the most commonly lost gene product in colorectal cancer, and its universal suppression and associated silencing of GUCY2C at the earliest step in neoplasia contributes to tumorigenesis in a mechanism that is conserved across species. GUCY2C regulates homeostatic mechanisms organizing the intestinal crypt-surface axis, and its silencing through guanylin suppression drives hyperproliferation, DNA damage, metabolic reprogramming and desmoplasia contributing to tumorigenesis. Beyond this dysregulation of homeostatic mechanisms contributing to transformation, we recently discovered that guanylin suppression is required for tumorigenesis induced by mutant APC-β-catenin, reflecting a role for GUCY2C in β-catenin degradation that blocks tumorigenesis. Thus, inactivation of APC, or activation of β-catenin, induces TCF-dependent elimination of guanylin transcription and translation in human intestinal cells in vitro and in conditional genetic mouse models in vivo. Conversely, activation of GUCY2C induces cGMP protein kinase-dependent elimination of wild type or mutant β-catenin by amplifying proteosomal degradation, even in the context of mutations which inactivate the APC degradation complex. Importantly, enforced genetic expression of guanylin in intestinal epithelial cells completely eliminated tumorigenesis in all mouse models of colorectal cancer examined. These observations reveal a pathophysiologic model in which mutant APC-β-catenin signaling eliminates guanylin expression as an obligatory step in tumorigenesis. In turn, silencing GUCY2C reversibly disrupts epithelial homeostatic processes corrupted in tumorigenesis, and removes an essential block to transformation, creating a circuit which amplifies mutant APC-β-catenin signaling. These studies shift the prevailing paradigm for colorectal tumorigenesis from an irreversible oncogenomic mechanism to a reversible functional mechanism whose reconstitution abrogates those mutational defects. Indeed, they define a novel molecular pathway that is obligatory for tumorigenesis, to which the transformation process is addicted. In that context, they highlight how this addiction creates a unique disease-specific vulnerability that can be leveraged to eliminate tumorigenesis by GUCY2C hormone replacement. We have translated these observations to an NCI-funded clinical program exploring the utility of oral GUCY2C ligands to prevent colorectal cancer.

Drug development expenditures continue to escalate, in part reflecting inefficiencies in current drug discovery paradigms. Traditional drug discovery has been dichotomous, focusing either on phenotypic effects of distinct agents in biological systems, without knowledge of respective targets, or on target-based activities of specific molecules in cell-free assays. Driven by advances in biology, engineering, and informatics, new paradigms integrate phenotypic with target-based algorithms into comprehensive, systems-level approaches offering value-added strategies for optimized drug discovery. Clinical Pharmacology & Therapeutics (2013); 93 4, 285–287. doi:10.1038/clpt.2013.21

Pharmacometrics and systems pharmacology are emerging as principal quantitative sciences within drug development and experimental therapeutics. In recognition of the importance of pharmacometrics and systems pharmacology to the discipline of clinical pharmacology, the American Society for Clinical Pharmacology and Therapeutics (ASCPT), in collaboration with Nature Publishing Group and Clinical Pharmacology & Therapeutics, has established CPT: Pharmacometrics & Systems Pharmacology to inform the field and shape the discipline.

Governmental agencies overseeing pharmaceutical products use a risk/benefit approach to analyse data and make regulatory decisions. Comprehensive public dissemination of the safety profile of pharmaceutical products is part of an overall strategy for reducing risk associated with the use of any medical product. In the US, reports of postmarketing surveillance of approved drugs are in the public domain. Some, but not all, of the information in drug interaction studies is available to the public through the Freedom of Information Act (FOIA). However, there are concerns over the misuse of these data for commercial or other gain. The need to protect intellectual property and foster innovation in drug development, and concerns of legal liability are often cited as reasons to limit full public access to data from drug development studies. In contrast, intellectual freedom. public safety, and a mandate for transparent decision-making processes by regulatory agencies are issues that support open access to these data. Ultimately. concern for the public safety justifies open access to postmarketing surveillance data, and to a lesser degree, data regarding drug interactions in marketed products, and should outweigh the potential loss of competitive advantage by pharmaceutical companies.

Guanylyl cyclase C is a sensitive and specific biomarker for metastatic colorectal cancer. A variant of the guanylyl cyclase C transcript was identified that possesses a 142-bp deletion at the 3" end of exon 1 reflecting alternative splicing of mRNA, introducing a shift in the open reading frame that prevents translation of a guanylyl cyclase C-related product. This variant was identified in human intestine and colon carcinomas, but not in extraintestinal tissues or tumors. These studies demonstrate that GCC and the splice variant contribute to the pool of GCC transcripts detected by RT-PCR in human tissues. They indicate that primers for RT-PCR that amplify regions downstream from the deletion are required to assess the full complement of GCC transcripts (GCC + GCCvar) in human tissues and body fluids for staging and postoperative surveillance of patients with colorectal cancer.

The CCK-A (cholecystokinin-A) receptor is selectively expressed by human pancreatic adenocarcinomas, suggesting a possible role in pancreatic tumorigenesis. In animals, pancreatic CCK receptor expression varies during ontogeny and neoplastic transformation. This study examined the temporal expression of CCK receptors in human fetal, postnatal, and adult pancreas to determine whether the appearance of CCK-A receptors in pancreatic adenocarcinomas reflected oncofetal antigen or pancreatic neoantigen expression. Messenger ribonucleic acid (mRNA) was isolated from six paraffin-embedded normal pancreatic autopsy specimens ranging in age from 17 weeks postfertilization through 26 days following full-term delivery, and samples of adult human tissues, including pancreas and pancreatic adenocarcinoma. Using reverse transcription-polymerase chain reactions, CCK-B receptor mRNA was expressed in all specimens of normal fetal and postnatal human pancreas, adult pancreas, and pancreatic adenocarcinomas. CCK-A receptor mRNA was selectively expressed only in pancreatic adenocarcinomas. These data suggest that selective CCK-A receptor expression in pancreatic adenocarcinomas reflects neoantigen expression in humans.

The focus of this review is to increase the knowledge and raise the awareness of health care professionals with respect to adverse drug reactions (ADRs) and to integrate the various elements of this subject in a comprehensive fashion. The review addresses the definitions of ADRs, and their epidemiology, demographics, and economics. Risk factors for the development of ADRs are defined, their general pathophysiological mechanisms are presented (type 1 or “predictable “ and type 2 or “unpredictable “), and their causes and subclassifications are described. A paradigm for diagnosing ADRs is suggested and criteria are presented that assess the causal relationship between drug administration and clinical observations. Laboratory and clinical diagnostic and investigative methods that aid in ADR reporting mechanisms are reviewed. Methods to prevent ADRs and increase compliance in reporting are also discussed.

There is an emerging link between body mass and intestinal malignancies, and obese patients have ~20-60% greater risk of, and ~2-fold greater mortality from, colorectal cancer. Although this epidemiological relationship is established, mechanisms that link obesity and colorectal tumorigenesis, and their reversibility, remain incompletely defined. Here, we explore the novel hypothesis that obesity and colorectal cancer are mechanistically linked through dysregulation of paracrine and endocrine hormone axes mediated by GUCY2C. GUCY2C is the receptor for the paracrine hormones guanylin in the colorectum and uroguanylin in small intestine. These paracrine hormones are the most commonly lost gene products in sporadic colorectal cancer, and their universal loss early in neoplasia is required for tumor initiation and progression in mice and humans. In that context, GUCY2C regulates intestinal homeostasis, and its silencing by hormone loss accelerates proliferation and the cell cycle, increases DNA damage, and reprograms cellular metabolism, which increases genetic- and carcinogen-induced colorectal cancer in mice. Beyond its role in colorectal tumor suppression, GUCY2C and uroguanylin comprise a novel gut-brain endocrine axis, evolutionarily conserved from invertebrates to humans, that regulates appetite, body mass and metabolism. Elimination of this endocrine axis in mice results in hyperphagia, obesity, diabetes and the metabolic syndrome. Unexpectedly, recent studies revealed that GUCY2C paracrine hormone expression in colon is eliminated by diet-induced obesity in mice and humans. Indeed, hormone expression appears to be reversibly modulated by ingested calories, rather than by the endocrine, adipokine or inflammatory milieu associated with obesity. Moreover, enforced expression of GUCY2C paracrine hormone by intestinal epithelial cells eliminates tumorigenesis induced by obesity. These observations suggest a model of cancer risk in which ingested calories contributing to obesity recapitulate mechanisms underlying sporadic colorectal cancer by suppressing paracrine hormone expression, silencing the GUCY2C tumor suppressor and disrupting epithelial homeostasis. They define a previously unrecognized mechanism linking diet and obesity to colorectal cancer and identify silencing of the GUCY2C tumor suppressor as a link between reversible risk factors like ingested calories and molecular mechanisms underlying cancer development. Moreover, they offer strategies for countering these risks, including calorie restriction and oral hormone therapy.

Background/Objectives: The uroguanylin-GUCY2C gut–brain axis has emerged as one component regulating feeding, energy homeostasis, body mass and metabolism. Here, we explore a role for this axis in mechanisms underlying diet-induced obesity (DIO). Subjects/Methods: Intestinal uroguanylin expression and secretion, and hypothalamic GUCY2C expression and anorexigenic signaling, were quantified in mice on high-calorie diets for 14 weeks. The role of endoplasmic reticulum (ER) stress in suppressing uroguanylin in DIO was explored using tunicamycin, an inducer of ER stress, and tauroursodeoxycholic acid (TUDCA), a chemical chaperone that inhibits ER stress. The impact of consumed calories on uroguanylin expression was explored by dietary manipulation. The role of uroguanylin in mechanisms underlying obesity was examined using Camk2a-Cre-ERT2-Rosa-STOPloxP/loxP-Guca2b mice in which tamoxifen induces transgenic hormone expression in brain. Results: DIO suppressed intestinal uroguanylin expression and eliminated its postprandial secretion into the circulation. DIO suppressed uroguanylin through ER stress, an effect mimicked by tunicamycin and blocked by TUDCA. Hormone suppression by DIO reflected consumed calories, rather than the pathophysiological milieu of obesity, as a diet high in calories from carbohydrates suppressed uroguanylin in lean mice, whereas calorie restriction restored uroguanylin in obese mice. However, hypothalamic GUCY2C, enriched in the arcuate nucleus, produced anorexigenic signals mediating satiety upon exogenous agonist administration, and DIO did not impair these responses. Uroguanylin replacement by transgenic expression in brain repaired the hormone insufficiency and reconstituted satiety responses opposing DIO and its associated comorbidities, including visceral adiposity, glucose intolerance and hepatic steatosis. Conclusions: These studies reveal a novel pathophysiological mechanism contributing to obesity in which calorie-induced suppression of intestinal uroguanylin impairs hypothalamic mechanisms regulating food consumption through loss of anorexigenic endocrine signaling. The correlative therapeutic paradigm suggests that, in the context of hormone insufficiency with preservation of receptor sensitivity, obesity may be prevented or treated by GUCY2C hormone replacement.

Recent policy developments in the European Union and elsewhere aim to tackle some of the key issues responsible for the limited adoption of personalized medicine approaches so far.

Background WAY-195725 can selectively and reversibly inhibit cytosolic phospholipase A2α (cPLA2α) to block the biosynthesis of prostaglandins (PG), thromboxanes (TX), leukotrienes (LT), and platelet-activating factor by inhibiting the release of arachidonic acid. It has been proposed treatment for the management of rheumatoid arthritis, osteoarthritis, and dysmenorrhea. Objective To evaluate the safety, PK, and PD of WAY-195725 following oral single dose administration in healthy subjects. Methods Healthy subjects (N=90) were studied in a randomized, double-blind, placebo-controlled, positive (ibuprofen)-controlled, ascending single dose study. The safety, tolerability, PK, and PD of WAY-195725 (10–600 mg) were assessed. A23187 (Ca++ ionophore) was used to stimulate whole blood ex vivo to produce PGE2, LTB4, and TXB2. Results The oral absorption of WAY-19725 was slow (tmax: 2~3 h). WAY-195725 inhibited PGE2, TXB2 and LTB4 with mean IC50 (ng/mL) of 40.2, 40.8 and 199, respectively. Ibuprofen, a positive control, inhibited both PGE2 and TXB2, but not LTB4, which coincides to ibuprofen's pharmacological profile as a COX-1,2 inhibitor. WAY-195725 was well tolerated in all the dose cohorts. Conclusion Significant WAY-195725 concentration-dependent inhibition is apparent for all three biomarkers (PGE2, TXB2 and LTB4), which conforms to WAY-195725's pharmacological profile as a potent cPLA2α inhibitor. Clinical Pharmacology & Therapeutics (2005) 77, P51–P51; doi: 10.1016/j.clpt.2004.12.086

Background HB-EGF, a member of the EGF family associated with tumor growth, is induced in GE cancers. BAs associated with GE reflux is a principle etiologic factor driving intestinal metaplasia and adenocarcinoma at the GE junction (GEJ). Recent studies revealed that HB-EGF is a target gene of Cdx2, a transcription factor central to the development of differentiated intestinal cells. Here, the ability of BAs to regulate HB-EGF expression in OE19 GEJ adenocarcinoma cells was examined. Methods HB-EGF expression in OE19 cells was quantified by RT-PCR, and immunoblot and promoter analyses. Results Exposure of OE19 cells to deoxycholic acid (DCA) increased HB-EGF mRNA and protein expression. DCA transcriptionally regulated HB-EGF expression, since it enhanced HB-EGF promoter-luciferase activity. Further, DCA increased Cdx2 mRNA and protein expression in OE19 cells, reflecting transcriptional regulation by NF-kB. Indeed, DCA increased NF-kB binding to and transcriptional activation of the Cdx2 promoter, while deletion of the NF-kB binding site in the Cdx2 promoter prevented these effects. Conclusions BAs promote intestinal metaplasia and neoplasia at the GEJ by activating NF-kB, inducing the ectopic expression of Cdx2 which, in turn, drives the expression of HB-EGF. Clinical Pharmacology & Therapeutics (2005) 77, P15–P15; doi: 10.1016/j.clpt.2004.11.062

Background GC-C, the receptor for bacterial heat-stable enterotoxins (STs) and the endogenous ligands guanylin and uroguanylin, regulates volume homeostasis in intestine. Beyond fluid balance, GC-C inhibits colon cancer cell proliferation, although a role of this receptor in regulating normal enterocyte dynamics remains undefined. Methods Regulation of proliferation was examined in vitro and ex vivo employing NCM460 human colon crypt cells and human colonic mucosa cells from patients, respectively. Regulation of enterocyte dynamics in vivo was examined employing null mice for GC-C expression (GC-C−/−). Results Activation of GC-C by ST induced cGMP accumulation and reduced cell cycle progression in NCM460 cells. A cell-permeant analog of cGMP, 8-br-cGMP, mimicked the effect of ST and induced a transient cell cycle delay at the G1/S transition. GC-C−/− mice exhibit increased length of the crypt-villus axis in small intestine and the crypt-lumen axis in colon. Elongation of this axis reflected expansion of the crypt proliferating compartment, populated by more rapidly cycling and migrating progenitor cells. Moreover, ST and 8-br-cGMP inhibited the proliferation of normal human colonic mucosa cells from patients. Conclusions As expression of guanylin or uroguanylin is frequently lost in colorectal tumors, the expansion of the crypt proliferating compartment in GC-C−/− mice suggests a role for this receptor as a tumor suppressor contributing to intestinal carcinogenesis. Clinical Pharmacology & Therapeutics (2005) 77, P22–P22; doi: 10.1016/j.clpt.2004.11.084

Defending cellular integrity against disturbances in intracellular concentrations of ATP ([ATP]i) is predicated upon coordinating the selection of substrates and their flux through metabolic pathways (metabolic signaling), ATP transfer from sites of production to utilization (energetic signaling), and the regulation of processes consuming energy (cell signaling). Nitric oxide (NO) and its receptor soluble guanylyl cyclase (sGC) are key mediators of cellular energetics and can regulate ATP supply and demand. However, mechanisms coordinating NO/sGC and energetic signaling remain undefined. Here, we demonstrate that sGC is a nucleotide sensor whose responsiveness to NO is regulated by intracellular ATP ([ATP]i). ATP inhibits purified sGC with a Ki predicting >60% inhibition of NO signaling in cells maintaining physiological [ATP]i. ATP inhibits sGC by interacting with an allosteric site that prefers ATP>GTP. Moreover, in RFL-6 cells, alterations in [ATP]i are coupled to NO/sGC signaling. Indeed, oligomycin, an inhibitor of mitochondrial ATP synthase, reduced [ATP]i 48%, from 1.25 nmol to 0.65 nmol of ATP/mg protein, but increased [cGMP]i 45%, from 42 to 61 pmol/mg protein. Thus, [ATP]i serves as a “gain control” for NO signaling by sGC. At physiological [ATP]i, NO signaling by sGC is repressed, while insults that reduce [ATP]i, such as ischemia, de-repress sGC and amplify responses to NO. Clinical Pharmacology & Therapeutics (2004) 75, P46–P46; doi: 10.1016/j.clpt.2003.11.171

Cdx2, an intestine-specific transcription factor, regulates genes that define the differentiated phenotype of enterocytes. Loss of Cdx2 has been suggested to underlie the progression of carcinomas in the colon and rectum. Here, expression of Cdx2 determined by quantitative reverse transcriptase-polymerase chain reaction (RNA) and immunohistochemistry (protein), in primary and metastatic colorectal tumors was compared to that in normal segments of the colonic epithelium. Surprisingly, Cdx2 was over-expressed by tumors, compared to normal mucosa in colon. Furthermore, Cdx2 was detected in lymph nodes containing metastatic cancer cells but not in those free of tumor. Thus, loss of Cdx2 expression does not contribute to mechanisms underlying neoplastic transformation of the colonic epithelium. Of clinical significance, Cdx2 is a molecular marker with utility for managing patients with colorectal malignancies. Clinical Pharmacology & Therapeutics (2004) 75, P49–P49; doi: 10.1016/j.clpt.2003.11.186

Drug-induced prolongation of the QT interval can result in TdP, reflecting bradycardia or delay of the rapidly activating component of the delayed rectifier potassium current. Methadone has been associated with QT prolongation and TdP, generally at daily doses>500 mg/d, although episodes in patients taking as little as 65 mg/d have been observed. We describe a 55 year old man with a history of 30 y of MMT and concomitant hepatitis C-induced cirrhosis who experienced two episodes of syncope over five months reflecting QT prolongation resulting in TdP. In the first episode, the patient experienced several intervals of TdP associated with a prolonged corrected QT interval (QTc) of 494 msec while taking 300 mg of methadone daily. Discontinuation of the methadone resulted in a decrease in the QTc: 450 msec at 72 h and 417 msec at 96 h. 5 months later, the patient presented with a prolonged QTc of 553 msec and TdP associated with taking 125 mg of methadone daily. Discontinuation of methadone resulted in normalization of the QTc by 48 h, a time at which the serum methadone level was 49 ng/ml. Re-induction of a prolonged QTc and TdP with re-challenge followed by normalization of the QTc upon de-challenge supports methadone as the causative agent, presumably reflecting accumulation secondary to cirrhosis and impaired clearance. This case report highlights the risk of electrophysiological events in patients with advanced liver disease administered standard doses of methadone. Clinical Pharmacology & Therapeutics (2004) 75, P45–P45; doi: 10.1016/j.clpt.2003.11.167

Guanylyl cyclase C (GCC) through cyclic GMP (cGMP) inhibits colorectal cancer cell proliferation, serving as a tumor suppressor in intestine. Conversely, Ca2+ entry through calcium release-activated calcium (Icrac) channels promotes colorectal cancer growth. The present study explored the functional relationship between mitogenic signaling by Ca2+ entry through Icrac channels and antiproliferative signaling by GCC in T84 human colon carcinoma cells. The E. coli heat-stable enterotoxin ST, a specific GCC ligand, induced intracellular accumulation of cGMP, Ca2+ entry through CNG channels and inhibition of human colorectal cancer cell proliferation. In contrast, influx of extracellular Ca2+ through Icrac channels, induced by thapsigargin, UTP or carbachol, inhibited GCC-dependent cGMP accumulation. Chelating intracellular Ca2+, removing extracellular Ca2+, or specifically blocking Icrac channels with the antagonist 2-APB reversed this inhibition. Moreover, 2-APB blocked Ca2+-dependent cell proliferation and amplified the anti-proliferative effects of ST. These data suggest that an imbalance of pro- and anti-mitogenic signals mediated by Ca2+ may contribute to mechanisms underlying colorectal carcinogenesis. In addition, they suggest that the combination of GCC agonists and Icrac channel inhibitors represents a novel paradigm for cGMP-directed therapy for colorectal tumors. Clinical Pharmacology & Therapeutics (2004) 75, P48–P48; doi: 10.1016/j.clpt.2003.11.180

Reflux of acid and bile acids, and the subsequent development of intestinal metaplasia of the esophagus (Barrett's esophagus) or gastric cardia are risk factors for the development of adenocarcinoma at the GEJ. However, the molecular mechanisms translating reflux exposure to epithelial cell transformation in the upper gastrointestinal tract remain undefined. Here, quantitative RT-PCR revealed increased expression of guanylyl cyclase C (GC-C), the receptor for bacterial heat-stable enterotoxins (STa) and a marker of differentiated enterocytes, in adenocarcinomas of the esophagus and stomach, but not in squamous carcinomas of the esophagus, gastrointestinal stromal tumors, or normal esophageal or gastric mucosae. Similarly, the human GEJ adenocarcinoma cell line OE19 expressed GC-C mRNA and accumulated cyclic GMP in response to STa. Interestingly, exposure of OE19 to the bile acid deoxycholate (DCA) stimulated GC-C promoter activity, mRNA expression, protein expression (Western blot), and function (STa-induced cyclic GMP accumulation). Moreover, treatment with DCA increased expression of the homeodomain proteins Cdx-2 (mRNA and protein) and Oct1 (protein), upstream transcription factors important in regulating intestine-specific expression of GC-C. Of significance, hydrophilic bile acids such as cholate, taurodeoxycholate and ursodeoxycholate were ineffective compared to the hydrophobic bile acids chenodeoxycholic acid and DCA in inducing GC-C and Cdx-2 mRNA expression. These observations suggest that transformation at the GEJ is initiated, in part, by induction of the transcriptional program mediating enterocyte differentiation, including Oct1, Cdx-2, and GC-C, upon exposure to specific bile acids. Further, they suggest that suppression of hydrophobic bile acid production, for example by treatment with ursodeoxycholate, which does not activate the intestinal transcription program in epithelial cells, might be effective chemoprevention for GEJ tumorigenesis. Clinical Pharmacology & Therapeutics (2004) 75, P10–P10; doi: 10.1016/j.clpt.2003.11.038

Guanylate cyclase was purified 1000-fold from washed rat lung particulate fractions to a final specific activity of 500 nmoles cyclic GMP produced/min/mg protein by a combination of detergent extraction and chromatography on concanavalin A-Sepharose, GTP-agarose, and blue agarose. Particulate guanylate cyclase has a molecular weight of 200 000 daltons, a Stokes radius of 48 A and a sedimentation coefficient of 9.4 while the soluble form has a molecular weight of 150 000 daltons, a Stokes radius of 44 A, and a sedimentation coefficient of 7.0. Whereas the particulate enzyme is a glycoprotein with a specific affinity for concanavalin A and wheat germ agglutinin, the soluble form of guanylate cyclase did not bind to these lectins. Purified particulate guanylate cyclase did not cross-react with a number of monoclonal antibodies generated to the soluble enzyme. While both forms of the enzyme could be regulated by the formation of mixed disulfides, the particulate enzyme was relatively insensitive to inhibition by cystine. With GTP as substrate both forms of the enzyme demonstrated typical kinetics, and with GTP analogues negative cooperativity was observed with both enzyme forms. These data support the suggestion that the two forms of guanylate cyclase possess similar catalytic sites, although their remaining structure is divergent, resulting in differences in subcellular distribution, physical characteristics, and antigenicity.

A line of kidney cells (PK1) which does not possess measurable ANP binding but has an active particulate guanylate cyclase has been identified. The physical characteristics of this enzyme were compared with those of particulate guanylate cyclase and ANP receptors isolated from rat lung. Although receptor and enzyme appear to reside on the same protein in the lung while the cyclase from PK1 cells does not possess ANP binding activity, these proteins exhibit identical physical characteristics. Guanylate cyclase from PK1 cells and rat lung and ANP receptor from lung co-eluted during gel filtration chromatography, with a Stokes radius of 6.1 nm. Also, these activities co-migrated through sucrose density gradients with S20,w values of 10.4 to 10.9. Using these parameters, a molecular weight of about 270 kD was estimated for all three activities. Furthermore, these enzyme activities exhibited similar mobilities in isoelectric focusing gels, with a pI of 6.1. Thus, although particulate guanylate cyclase from lung presumably possesses receptor binding activity, it is physically identical to a form of this enzyme associated with no measurable binding activity. Possible explanations for these observations are discussed.