An engineered bacterial therapeutic lowers urinary oxalate in preclinical models and in silico simulations of enteric hyperoxaluria.

Enteric hyperoxaluria (EH) is a metabolic disease caused by excessive absorption of dietary oxalate leading to the formation of chronic kidney stones and kidney failure. There are no approved pharmaceutical treatments for EH. SYNB8802 is an engineered bacterial therapeutic designed to consume oxalate in the gut and lower urinary oxalate as a potential treatment for EH. Oral administration of SYNB8802 leads to significantly decreased urinary oxalate excretion in healthy mice and non-human primates, demonstrating the strain's ability to consume oxalate in vivo. A mathematical modeling framework was constructed that combines in vitro and in vivo preclinical data to predict the effects of SYNB8802 administration on urinary oxalate excretion in humans. Simulations of SYNB8802 administration predict a clinically meaningful lowering of urinary oxalate excretion in healthy volunteers and EH patients. Together, these findings suggest that SYNB8802 is a promising treatment for EH.

Genetically engineered E. coli Nissle attenuates hyperammonemia and prevents memory impairment in bile-duct ligated rats.

Hyperammonemia associated with chronic liver disease (CLD) is implicated in the pathogenesis of hepatic encephalopathy (HE). The gut is a major source of ammonia production that contributes to hyperammonemia in CLD and HE and remains the primary therapeutic target for lowering hyperammonemia. As an ammonia-lowering strategy, Escherichia coli Nissle 1917 bacterium was genetically modified to consume and convert ammonia to arginine (S-ARG). S-ARG was further modified to additionally synthesize butyrate (S-ARG + BUT). Both strains were evaluated in bile-duct ligated (BDL) rats; experimental model of CLD and HE. METHODS: One-week post-surgery, BDLs received non-modified EcN (EcN), S-ARG, S-ARG + BUT (3x1011 CFU/day) or vehicle until sacrifice at 3 or 5 weeks. Plasma (ammonia/pro-inflammatory/liver function), liver fibrosis (hydroxyproline), liver mRNA (pro-inflammatory/fibrogenic/anti-apoptotic) and colon mRNA (pro-inflammatory) biomarkers were measured post-sacrifice. Memory, motor-coordination, muscle-strength and locomotion were assessed at 5 weeks. RESULTS: In BDL-Veh rats, hyperammonemia developed at 3 and further increased at 5 weeks. This rise was prevented by S-ARG and S-ARG + BUT, whereas EcN was ineffective. Memory impairment was prevented only in S-ARG + BUT vs BDL-Veh. Systemic inflammation (IL-10/MCP-1/endotoxin) increased at 3 and 5 weeks in BDL-Veh. S-ARG + BUT attenuated inflammation at both timepoints (except 5-week endotoxin) vs BDL-Veh, whereas S-ARG only attenuated IP-10 and MCP-1 at 3 weeks. Circulating ALT/AST/ALP/GGT/albumin/bilirubin and gene expression of liver function markers (IL-10/IL-6/IL-1ß/TGF-ß/α-SMA/collagen-1α1/Bcl-2) were not normalized by either strain. Colonic mRNA (TNF-α/IL-1ß/occludin) markers were attenuated by synthetic strains at both timepoints vs BDL-Veh. CONCLUSION: S-ARG and S-ARG + BUT attenuated hyperammonemia, with S-ARG + BUT additional memory protection likely due to greater anti-inflammatory effect. These innovative strategies, particularly S-ARG + BUT, have potential to prevent HE.

Low-Dose Linaclotide (72 µg) for Chronic Idiopathic Constipation: A 12-Week, Randomized, Double-Blind, Placebo-Controlled Trial.

OBJECTIVES: Linaclotide is a guanylate cyclase-C agonist approved in the United States, Canada, and Mexico at a once-daily 145-µg dose for the treatment of chronic idiopathic constipation (CIC); a once-daily 72-µg dose for CIC recently received FDA approval. The trial objective was to evaluate the efficacy and safety of a 72-µg linaclotide dose in CIC patients. METHODS: This double-blind, placebo-controlled trial randomized patients with CIC (Rome III criteria) to once-daily linaclotide 72 µg or 145 µg, or placebo for 12 weeks. The primary endpoint, 12-week complete spontaneous bowel movement (CSBM) overall responder, required patients to have ≥3 CSBMs and an increase of ≥1 CSBM per week from baseline in the same week for ≥9 of 12 weeks of the treatment period. Secondary endpoints included 12-week change from baseline in bowel (SBM and CSBM frequency, stool consistency, straining) and abdominal (bloating, discomfort) symptoms, monthly CSBM responders, and 12-week CSBM responders among patients who averaged >1 SBM/week at baseline. Sustained response (12-week CSBM overall responders who met weekly criteria for 3 of the 4 final weeks (weeks 9-12) of treatment) was evaluated as an additional endpoint. Adverse events (AEs) were monitored. RESULTS: The intent-to-treat population included 1,223 patients (mean age=46 years, female=77%, white=71%). The primary endpoint was met by 13.4% of linaclotide 72-µg patients vs. 4.7% of placebo patients (P<0.0001, odds ratio=3.0; statistically significant controlling for multiplicity). Sustained response was achieved by 12.4% of linaclotide 72-µg patients vs. 4.2% of placebo patients (nominal P<0.0001). Linaclotide 72-µg patients met 9-of-10 secondary endpoints vs. placebo (P<0.05; abdominal discomfort, P=0.1028). Patients treated with linaclotide 145 µg also improved CIC symptoms for the primary (12.4%) and sustained responder endpoint parameters (11.4%) and for all 10 of the secondary endpoint parameters including abdominal discomfort (P<0.05). Diarrhea, the most common AE, was mild in most instances and resulted in discontinuation of 0, 2.4%, and 3.2% of patients in the placebo, linaclotide 72-µg, and linaclotide 145-µg groups, respectively. CONCLUSIONS: Once-daily linaclotide 72 µg significantly improved CIC symptoms in both men and women with a low rate of discontinuation due to diarrhea over 12 weeks of treatment.

MRP4 Modulation of the Guanylate Cyclase-C/cGMP Pathway: Effects on Linaclotide-Induced Electrolyte Secretion and cGMP Efflux.

MRP4 mediates the efflux of cGMP and cAMP and acts as an important regulator of these secondary messengers, thereby affecting signaling events mediated by cGMP and cAMP. Immunofluorescence staining showed high MRP4 expression localized predominantly in the apical membrane of rat colonic epithelium. In vitro studies were performed using a rat colonic mucosal layer mounted in an Ussing chamber. Linaclotide activation of the guanylate cyclase-C (GC-C)/cGMP pathway induced a concentration-dependent increase in transepithelial ion current [short-circuit current (Isc)] across rat colonic mucosa (EC50: 9.2 nM). Pretreatment of colonic mucosa with the specific MRP4 inhibitor MK571 potentiated linaclotide-induced electrolyte secretion and augmented linaclotide-stimulated intracellular cGMP accumulation. Notably, pretreatment with the phosphodiesterase 5 inhibitor sildenafil increased basal Isc, but had no amplifying effect on linaclotide-induced Isc. MRP4 inhibition selectively affected the activation phase, but not the deactivation phase, of linaclotide. In contrast, incubation with a GC-C/Fc chimera binding to linaclotide abrogated linaclotide-induced Isc, returning to baseline. Furthermore, linaclotide activation of GC-C induced cGMP secretion from the apical and basolateral membranes of colonic epithelium. MRP4 inhibition blocked cGMP efflux from the apical membrane, but not the basolateral membrane. These data reveal a novel, previously unrecognized mechanism that functionally couples GC-C-induced luminal electrolyte transport and cGMP secretion to spatially restricted, compartmentalized regulation by MRP4 at the apical membrane of intestinal epithelium. These findings have important implications for gastrointestinal disorders with symptoms associated with dysregulated fluid homeostasis, such as irritable bowel syndrome with constipation, chronic idiopathic constipation, and secretory diarrhea.

Two randomized trials of linaclotide for chronic constipation.

BACKGROUND: Linaclotide is a minimally absorbed peptide agonist of the guanylate cyclase C receptor. In two trials, we aimed to determine the efficacy and safety of linaclotide in patients with chronic constipation. METHODS: We conducted two randomized, 12-week, multicenter, double-blind, parallel-group, placebo-controlled, dual-dose trials (Trials 303 and 01) involving 1276 patients with chronic constipation. Patients received either placebo or linaclotide, 145 µg or 290 µg, once daily for 12 weeks. The primary efficacy end point was three or more complete spontaneous bowel movements (CSBMs) per week and an increase of one or more CSBMs from baseline during at least 9 of the 12 weeks. Adverse events were also monitored. RESULTS: For Trials 303 and 01, respectively, the primary end point was reached by 21.2% and 16.0% of the patients who received 145 µg of linaclotide and by 19.4% and 21.3% of the patients who received 290 µg of linaclotide, as compared with 3.3% and 6.0% of those who received placebo (P<0.01 for all comparisons of linaclotide with placebo). Improvements in all secondary end points were significantly greater in both linaclotide groups than in the placebo groups. The incidence of adverse events was similar among all study groups, with the exception of diarrhea, which led to discontinuation of treatment in 4.2% of patients in both linaclotide groups. CONCLUSIONS: In these two 12-week trials, linaclotide significantly reduced bowel and abdominal symptoms in patients with chronic constipation. Additional studies are needed to evaluate the potential long-term risks and benefits of linaclotide in chronic constipation. (Funded by Ironwood Pharmaceuticals and Forest Research Institute; ClinicalTrials.gov numbers, NCT00765882 and NCT00730015.).

Effect of linaclotide on severe abdominal symptoms in patients with irritable bowel syndrome with constipation.

BACKGROUND & AIMS: Patients with irritable bowel syndrome with constipation (IBS-C) have abdominal symptoms that vary in severity. Linaclotide, a guanylate cyclase-C agonist, improves abdominal and bowel symptoms in these patients. We examined the prevalence of severe abdominal symptoms in patients with IBS-C and assessed the effects of linaclotide on abdominal symptoms, global measures, and quality of life (QOL). METHODS: In two phase 3 trials, patients who met modified Rome II criteria for IBS-C were randomly assigned to groups given oral, once-daily linaclotide (290 µg) or placebo for 12 weeks. During the baseline (2 weeks prior to treatment) and treatment periods, patients rated abdominal pain, discomfort, bloating, fullness, and cramping daily (from 0 = none to 10 = very severe). Linaclotide's effects on abdominal symptoms, global measures, and IBS-related QOL were assessed in subpopulations of patients who rated specific individual abdominal symptoms as severe (≥ 7.0) at baseline. RESULTS: In the intent-to-treat population (1602 patients; 797 receiving placebo and 805 receiving linaclotide), baseline prevalence values for severe abdominal symptoms were 44% for bloating, 44% for fullness, 32% for discomfort, 23% for pain, and 22% for cramping, with considerable overlap among symptoms. In patients with severe symptoms, linaclotide reduced all abdominal symptoms; mean changes from baseline severity scores ranged from -2.7 to -3.4 for linaclotide vs -1.4 to -1.9 for placebo (P < .0001). Linaclotide improved global measures (P < .0001) and IBS-QOL scores (P < .01) compared with placebo. Diarrhea was the most common adverse event of linaclotide in patients with severe abdominal symptoms (18.8%-21.0%). CONCLUSIONS: Of 5 severe abdominal symptoms assessed, bloating and fullness were most prevalent in patients with IBS-C. Linaclotide significantly improved all abdominal symptoms, global measures, and IBS-QOL in subpopulations of IBS-C patients with severe abdominal symptoms. Clinicaltrials.gov NUMBERS: NCT00938717, NCT00948818.

Linaclotide inhibits colonic nociceptors and relieves abdominal pain via guanylate cyclase-C and extracellular cyclic guanosine 3',5'-monophosphate.

BACKGROUND & AIMS: Linaclotide is a minimally absorbed agonist of guanylate cyclase-C (GUCY2C or GC-C) that reduces symptoms associated with irritable bowel syndrome with constipation (IBS-C). Little is known about the mechanism by which linaclotide reduces abdominal pain in patients with IBS-C. METHODS: We determined the effects of linaclotide on colonic sensory afferents in healthy mice and those with chronic visceral hypersensitivity. We assessed pain transmission by measuring activation of dorsal horn neurons in the spinal cord in response to noxious colorectal distention. Levels of Gucy2c messenger RNA were measured in tissues from mice using quantitative reverse transcription polymerase chain reaction and in situ hybridization. We used human intestinal cell lines to measure release of cyclic guanosine-3',5'-monophosphate (cGMP) by linaclotide. We performed a post-hoc analysis of data from a phase III, double-blind, parallel-group study in which 805 patients with IBS-C were randomly assigned to groups given an oral placebo or 290 µg linaclotide once daily for 26 weeks. We quantified changes in IBS-C symptoms, including abdominal pain. RESULTS: In mice, linaclotide inhibited colonic nociceptors with greater efficacy during chronic visceral hypersensitivity. Intra-colonic administration of linaclotide reduced signaling of noxious colorectal distention to the spinal cord. The colonic mucosa, but not neurons, was found to express linaclotide's target, GC-C. The downstream effector of GC-C, cGMP, was released after administration of linaclotide and also inhibited nociceptors. The effects of linaclotide were lost in Gucy2c(-/-) mice and prevented by inhibiting cGMP transporters or removing the mucosa. During 26 weeks of linaclotide administration, a significantly greater percentage of patients (70%) had at least a 30% reduction in abdominal pain compared with patients given placebo (50%). CONCLUSIONS: We have identified an analgesic mechanism of linaclotide: it activates GC-C expressed on mucosal epithelial cells, resulting in the production and release of cGMP. This extracellular cGMP acts on and inhibits nociceptors, thereby reducing nociception. We also found that linaclotide reduces chronic abdominal pain in patients with IBS-C.

Pharmacologic properties, metabolism, and disposition of linaclotide, a novel therapeutic peptide approved for the treatment of irritable bowel syndrome with constipation and chronic idiopathic constipation.

Linaclotide, a potent guanylate cyclase C agonist, is a therapeutic peptide approved in the United States for the treatment of irritable bowel syndrome with constipation and chronic idiopathic constipation. We present for the first time the metabolism, degradation, and disposition of linaclotide in animals and humans. We examined the metabolic stability of linaclotide in conditions that mimic the gastrointestinal tract and characterized the metabolite MM-419447 (CCEYCCNPACTGC), which contributes to the pharmacologic effects of linaclotide. Systemic exposure to these active peptides is low in rats and humans, and the low systemic and portal vein concentrations of linaclotide and MM-419447 observed in the rat confirmed both peptides are minimally absorbed after oral administration. Linaclotide is stable in the acidic environment of the stomach and is converted to MM-419447 in the small intestine. The disulfide bonds of both peptides are reduced in the small intestine, where they are subsequently proteolyzed and degraded. After oral administration of linaclotide, <1% of the dose was excreted as active peptide in rat feces and a mean of 3-5% in human feces; in both cases MM-419447 was the predominant peptide recovered. MM-419447 exhibits high-affinity binding in vitro to T84 cells, resulting in a significant, concentration-dependent accumulation of intracellular cyclic guanosine-3',5'-monophosphate (cGMP). In rat models of gastrointestinal function, orally dosed MM-419447 significantly increased fluid secretion into small intestinal loops, increased intraluminal cGMP, and caused a dose-dependent acceleration in gastrointestinal transit. These results demonstrate the importance of the active metabolite in contributing to linaclotide's pharmacology.

Linaclotide for irritable bowel syndrome with constipation: a 26-week, randomized, double-blind, placebo-controlled trial to evaluate efficacy and safety.

OBJECTIVES: Linaclotide is a minimally absorbed peptide guanylate cyclase-C agonist. The objective of this trial was to determine the efficacy and safety of linaclotide treatment in patients with irritable bowel syndrome with constipation (IBS-C) over 26 weeks. METHODS: This phase 3, double-blind, parallel-group, placebo-controlled trial randomized IBS-C patients to placebo or 290 µg of oral linaclotide once daily for a 26-week treatment period. The primary and the secondary efficacy assessments were evaluated over the first 12 weeks of treatment. Primary end points included the Food and Drug Administration's (FDA's) end point for IBS-C (responder: a patient who reported (i) improvement of ≥ 30 % from baseline in average daily worst abdominal pain score and (ii) increase of ≥ 1 complete spontaneous bowel movement (CSBM) from baseline, both in the same week for ≥ 6 / 12 weeks) and three other primary end points, based on improvements in abdominal pain and CSBMs for 9/12 weeks. Adverse events (AEs) were monitored. RESULTS: In all, 804 patients (mean age = 44 years, female = 90 % , white = 78 % ) were evaluated; 33.7 % of linaclotide-treated patients were FDA end point responders, vs. 13.9 % of placebo-treated patients ( P < 0.0001) (number needed to treat = 5.1, 95 % confidence interval (CI): 3.9, 7.1). The pain responder criterion of the FDA end point was met by 48.9 % of linaclotide-treated patients vs. 34.5 % of placebo-treated patients (number needed to treat = 7.0, 95 % CI: 4.7, 13.1), and the CSBM responder criterion was met by 47.6 % of linaclotide-treated patients, vs. 22.6 % of placebo patients (number needed to treat = 4.0, 95 % CI: 3.2, 5.4). Remaining primary end points ( P < 0.0001) and all secondary end points ( P < 0.001), including abdominal pain, abdominal bloating, and bowel symptoms (SBM and CSBM rates, Bristol Stool Form Scale (BSFS) score, and straining), were also statistically significantly improved with linaclotide vs. placebo. Statistically significant differences from placebo were observed for responder and continuous end points over 26 weeks of treatment. AE incidence was similar between treatment groups, except for diarrhea, which caused discontinuation in 4.5 % of linaclotide patients vs. 0.2 % of placebo patients. CONCLUSIONS: Linaclotide 290 µg once daily significantly improved abdominal and bowel symptoms associated with IBS-C over 26 weeks of treatment.

A 12-week, randomized, controlled trial with a 4-week randomized withdrawal period to evaluate the efficacy and safety of linaclotide in irritable bowel syndrome with constipation.

OBJECTIVES: Linaclotide is a minimally absorbed guanylate cyclase-C agonist. The objective of this trial was to determine the efficacy and safety of linaclotide in patients with irritable bowel syndrome with constipation (IBS-C). METHODS: This phase 3, double-blind, parallel-group, placebo-controlled trial randomized IBS-C patients to placebo or 290 µ g oral linaclotide once daily in a 12-week treatment period, followed by a 4-week randomized withdrawal (RW) period. There were four primary end points, the Food and Drug Administration ' s (FDA ' s) primary end point for IBS-C (responder: improvement of ≥ 30 % in average daily worst abdominal pain score and increase by ≥ 1 complete spontaneous bowel movement (CSBM) from baseline (same week) for at least 50 % of weeks assessed) and three other primary end points, based on improvements in abdominal pain and CSBMs for 9 / 12 weeks. Adverse events (AEs) were monitored. RESULTS: The trial evaluated 800 patients (mean age = 43.5 years, female = 90.5 % , white = 76.9 % ). The FDA end point was met by 136 / 405 linaclotide-treated patients (33.6 % ), compared with 83 / 395 placebo-treated patients (21.0 % ) ( P < 0.0001) (number needed to treat: 8.0, 95 % confidence interval: 5.4, 15.5). A greater percentage of linaclotide patients, compared with placebo patients, reported for at least 6 / 12 treatment period weeks, a reduction of ≥ 30 % in abdominal pain (50.1 vs. 37.5 % , P = 0.0003) and an increase of ≥ 1 CSBM from baseline (48.6 vs. 29.6 % , P < 0.0001). A greater percentage of linaclotide patients vs. placebo patients were also responders for the other three primary end points ( P < 0.05). Significantly greater improvements were seen in linaclotide vs. placebo patients for all secondary end points ( P < 0.001). During the RW period, patients remaining on linaclotide showed sustained improvement; patients re-randomized from linaclotide to placebo showed return of symptoms, but without worsening of symptoms relative to baseline. Diarrhea, the most common AE, resulted in discontinuation of 5.7 % of linaclotide and 0.3 % of placebo patients. CONCLUSIONS: Linaclotide significantly improved abdominal pain and bowel symptoms associated with IBS-C for at least 12 weeks; there was no worsening of symptoms compared with baseline following cessation of linaclotide during the RW period.

Efficacy of linaclotide for patients with chronic constipation.

BACKGROUND & AIMS: Linaclotide is a minimally absorbed peptide agonist of the guanylate cyclase-C receptor that stimulates intestinal fluid secretion and transit and reduces pain in animal models. We assessed the safety and efficacy of a range of linaclotide doses in patients with chronic constipation. METHODS: We performed a multicenter, double-blind, placebo-controlled, parallel-group study of 310 patients with chronic constipation. Patients were randomly assigned to groups given 75, 150, 300, or 600 microg oral linaclotide or placebo once daily for 4 weeks. Symptom assessments included spontaneous bowel movements (SBMs), complete SBMs, stool consistency, straining, abdominal discomfort, and bloating. Severity of constipation, adequate relief of constipation, global relief of constipation, treatment satisfaction, quality of life, adverse events, clinical laboratory data, and electrocardiogram results were assessed. RESULTS: All doses of linaclotide improved the weekly rate of SBM (primary end point) compared with placebo; the increases in overall weekly number of SBMs from baseline were 2.6, 3.3, 3.6, and 4.3 for linaclotide doses of 75, 150, 300, and 600 microg, respectively, compared with 1.5 for placebo (P < or = .05 for each pair-wise comparison of a linaclotide dose to placebo). Likewise, linaclotide significantly improved the weekly rate of complete SBM, stool consistency, straining, abdominal discomfort, bloating, global assessments, and quality of life. The most common and only dose-related adverse event was diarrhea (only 6 patients discontinued treatment because of diarrhea). CONCLUSIONS: Linaclotide therapy was associated with few adverse events and produced rapid and sustained improvement of bowel habits, abdominal symptoms, global relief, and quality of life in patients with chronic constipation.

Linaclotide improves abdominal pain and bowel habits in a phase IIb study of patients with irritable bowel syndrome with constipation.

BACKGROUND & AIMS: Linaclotide, a minimally absorbed, 14-amino acid peptide agonist of guanylate cyclase-C, has shown benefit in a proof-of-concept study for the treatment of patients with irritable bowel syndrome (IBS) with constipation (IBS-C). We assessed the efficacy and safety of linaclotide at a daily dose range of 75-600 µg in IBS-C. METHODS: We performed a randomized, double-blind, multicenter, placebo-controlled study of 420 patients with IBS-C given oral linaclotide at doses of 75, 150, 300, or 600 µg or placebo once daily for 12 weeks. End points included change from baseline in daily bowel habits, daily abdominal symptoms, and weekly global assessments, in addition to responder criteria. RESULTS: All doses of linaclotide significantly improved bowel habits, including frequency of spontaneous bowel movements and complete spontaneous bowel movements (primary end point), severity of straining, and stool consistency. Abdominal pain was significantly reduced from baseline, compared with placebo; mean changes in abdominal pain (assessed on a 5-point scale) from baseline were -0.71, -0.71, -0.90, and -0.86 for linaclotide doses of 75, 150, 300, and 600 µg, respectively, compared with -0.49 for placebo. Likewise, most doses of linaclotide significantly improved other abdominal symptoms, including discomfort and bloating, and global measures of IBS-C compared with placebo. Effects were observed within the first week and were sustained throughout 12 weeks of treatment. Except for diarrhea, the incidence of adverse events was similar between placebo and linaclotide groups. CONCLUSIONS: Linaclotide, across a wide range of doses, significantly improved symptoms of IBS-C, including abdominal pain and bowel symptoms. Diarrhea was the only dose-dependent adverse event and was usually of mild or moderate severity.

Safety and pharmacodynamics of an engineered E. coli Nissle for the treatment of phenylketonuria: a first-in-human phase 1/2a study.

Phenylketonuria (PKU) is a rare disease caused by biallelic mutations in the PAH gene that result in an inability to convert phenylalanine (Phe) to tyrosine, elevated blood Phe levels and severe neurological complications if untreated. Most patients are unable to adhere to the protein-restricted diet, and thus do not achieve target blood Phe levels. We engineered a strain of E. coli Nissle 1917, designated SYNB1618, through insertion of the genes encoding phenylalanine ammonia lyase and L-amino acid deaminase into the genome, which allow for bacterial consumption of Phe within the gastrointestinal tract. SYNB1618 was studied in a phase 1/2a randomized, placebo-controlled, double-blind, multi-centre, in-patient study ( NCT03516487 ) in adult healthy volunteers (n = 56) and patients with PKU and blood Phe level ≥600 mmol l-1 (n = 14). Participants were randomized to receive a single dose of SYNB1618 or placebo (part 1) or up to three times per day for up to 7 days (part 2). The primary outcome of this study was safety and tolerability, and the secondary outcome was microbial kinetics. A D5-Phe tracer (15 mg kg-1) was used to study exploratory pharmacodynamic effects. SYNB1618 was safe and well tolerated with a maximum tolerated dose of 2 × 1011 colony-forming units. Adverse events were mostly gastrointestinal and of mild to moderate severity. All participants cleared the bacteria within 4 days of the last dose. Dose-responsive increases in strain-specific Phe metabolites in plasma (trans-cinnamic acid) and urine (hippuric acid) were observed, providing a proof of mechanism for the potential to use engineered bacteria in the treatment of rare metabolic disorders.

Developing a new class of engineered live bacterial therapeutics to treat human diseases.

A complex interplay of metabolic and immunological mechanisms underlies many diseases that represent a substantial unmet medical need. There is an increasing appreciation of the role microbes play in human health and disease, and evidence is accumulating that a new class of live biotherapeutics comprised of engineered microbes could address specific mechanisms of disease. Using the tools of synthetic biology, nonpathogenic bacteria can be designed to sense and respond to environmental signals in order to consume harmful compounds and deliver therapeutic effectors. In this perspective, we describe considerations for the design and development of engineered live biotherapeutics to achieve regulatory and patient acceptance.

Pilot study on the effect of linaclotide in patients with chronic constipation.

OBJECTIVES: Chronic constipation is a common gastrointestinal disorder with limited treatment options. Oral administration of linaclotide, a novel peptide agonist of guanylate cyclase-C receptors, has been shown in animal studies to stimulate intestinal fluid secretion and transit. In Phase 1 studies in healthy human volunteers, linaclotide was well-tolerated, increased bowel movement frequency, and loosened stool consistency. METHODS: This randomized, double-blind, placebo-controlled pilot study evaluated the safety, tolerability, and exploratory efficacy of oral linaclotide in 42 patients with chronic constipation. Patients were randomized to linaclotide (100, 300, or 1,000 microg) or placebo once daily for 2 weeks. Bowel habits (stool frequency, consistency, straining, completeness of evacuation) and degree of abdominal discomfort were monitored daily using an interactive voice response system. Patient-reported outcomes of severity of constipation and overall relief were evaluated weekly. RESULTS: Linaclotide treatment produced dose-dependent increases from the pretreatment baseline values in weekly complete spontaneous bowel movement frequency (range: 2.2-3.2), stool consistency scores (range: 1.1-2.6, 7-point scale), and straining scores (range: 0.4-1.5, 7-point scale); corresponding placebo increases were 1.3, 0.4, 0.4, respectively. Clinical improvements were also demonstrated in abdominal discomfort, severity of constipation, and overall relief. Compared to placebo, linaclotide 100 microg/day significantly increased spontaneous bowel movement frequency, and linaclotide 1,000 microg/day significantly improved stool consistency (P<0.05). Adverse events were primarily gastrointestinal, with diarrhea being the most common. CONCLUSIONS: In this pilot study, linaclotide treatment improved bowel habits and symptoms of patients with chronic constipation. Further randomized controlled trials are warranted.

An engineered E. coli Nissle improves hyperammonemia and survival in mice and shows dose-dependent exposure in healthy humans.

The intestine is a major source of systemic ammonia (NH3); thus, capturing part of gut NH3 may mitigate disease symptoms in conditions of hyperammonemia such as urea cycle disorders and hepatic encephalopathy. As an approach to the lowering of blood ammonia arising from the intestine, we engineered the orally delivered probiotic Escherichia coli Nissle 1917 to create strain SYNB1020 that converts NH3 to l-arginine (l-arg). We up-regulated arginine biosynthesis in SYNB1020 by deleting a negative regulator of l-arg biosynthesis and inserting a feedback-resistant l-arg biosynthetic enzyme. SYNB1020 produced l-arg and consumed NH3 in an in vitro system. SYNB1020 reduced systemic hyperammonemia, improved survival in ornithine transcarbamylase-deficient spfash mice, and decreased hyperammonemia in the thioacetamide-induced liver injury mouse model. A phase 1 clinical study was conducted including 52 male and female healthy adult volunteers. SYNB1020 was well tolerated at daily doses of up to 1.5 × 1012 colony-forming units administered for up to 14 days. A statistically significant dose-dependent increase in urinary nitrate, plasma 15N-nitrate (highest dose versus placebo, P = 0.0015), and urinary 15N-nitrate was demonstrated, indicating in vivo SYNB1020 activity. SYNB1020 concentrations reached steady state by the second day of dosing, and excreted cells were alive and metabolically active as evidenced by fecal arginine production in response to added ammonium chloride. SYNB1020 was no longer detectable in feces 2 weeks after the last dose. These results support further clinical development of SYNB1020 for hyperammonemia disorders including urea cycle disorders and hepatic encephalopathy.

Chronic linaclotide treatment reduces colitis-induced neuroplasticity and reverses persistent bladder dysfunction.

Irritable bowel syndrome (IBS) patients suffer from chronic abdominal pain and extraintestinal comorbidities, including overactive bladder (OAB) and interstitial cystitis/painful bladder syndrome (IC-PBS). Mechanistic understanding of the cause and time course of these comorbid symptoms is lacking, as are clinical treatments. Here, we report that colitis triggers hypersensitivity of colonic afferents, neuroplasticity of spinal cord circuits, and chronic abdominal pain, which persists after inflammation. Subsequently, and in the absence of bladder pathology, colonic hypersensitivity induces persistent hypersensitivity of bladder afferent pathways, resulting in bladder-voiding dysfunction, indicative of OAB/IC-PBS. Daily administration of linaclotide, a guanylate cyclase-C (GC-C) agonist that is restricted to and acts within the gastrointestinal tract, reverses colonic afferent hypersensitivity, reverses neuroplasticity-induced alterations in spinal circuitry, and alleviates chronic abdominal pain in mice. Intriguingly, daily linaclotide administration also reverses persistent bladder afferent hypersensitivity to mechanical and chemical stimuli and restores normal bladder voiding. Linaclotide itself does not inhibit bladder afferents, rather normalization of bladder function by daily linaclotide treatment occurs via indirect inhibition of bladder afferents via reduced nociceptive signaling from the colon. These data support the concepts that cross-organ sensitization underlies the development and maintenance of visceral comorbidities, while pharmaceutical treatments that inhibit colonic afferents may also improve urological symptoms through common sensory pathways.

Development of a synthetic live bacterial therapeutic for the human metabolic disease phenylketonuria.

Phenylketonuria (PKU) is a genetic disease that is characterized by an inability to metabolize phenylalanine (Phe), which can result in neurotoxicity. To provide a potential alternative to a protein-restricted diet, we engineered Escherichia coli Nissle to express genes encoding Phe-metabolizing enzymes in response to anoxic conditions in the mammalian gut. Administration of our synthetic strain, SYNB1618, to the Pahenu2/enu2 PKU mouse model reduced blood Phe concentration by 38% compared with the control, independent of dietary protein intake. In healthy Cynomolgus monkeys, we found that SYNB1618 inhibited increases in serum Phe after an oral Phe dietary challenge. In mice and primates, Phe was converted to trans-cinnamate by SYNB1618, quantitatively metabolized by the host to hippurate and excreted in the urine, acting as a predictive biomarker for strain activity. SYNB1618 was detectable in murine or primate feces after a single oral dose, permitting the evaluation of pharmacodynamic properties. Our results define a strategy for translation of live bacterial therapeutics to treat metabolic disorders.

Guanylate cyclase 2C agonism corrects CFTR mutants.

Cystic fibrosis (CF) is a genetic disorder in which epithelium-generated fluid flow from the lung, intestine, and pancreas is impaired due to mutations disrupting CF transmembrane conductance regulator (CFTR) channel function. CF manifestations of the pancreas and lung are present in the vast majority of CF patients, and 15% of CF infants are born with obstructed gut or meconium ileus. However, constipation is a significantly underreported outcome of CF disease, affecting 47% of the CF patients, and management becomes critical in the wake of increasing life span of CF patients. In this study, we unraveled a potentially novel molecular role of a membrane-bound cyclic guanosine monophosphate-synthesizing (cGMP-synthesizing) intestinal enzyme, guanylate cyclase 2C (GCC) that could be targeted to ameliorate CF-associated intestinal fluid deficit. We demonstrated that GCC agonism results in functional rescue of murine F508del/F508del and R117H/R117H Cftr and CFTR mutants in CF patient-derived intestinal spheres. GCC coexpression and activation facilitated processing and ER exit of F508del CFTR and presented a potentially novel rescue modality in the intestine, similar to the CF corrector VX-809. Our findings identify GCC as a biological CFTR corrector and potentiator in the intestine.