Development of a mechanistic model to predict synthetic biotic activity in healthy volunteers and patients with phenylketonuria.

The development of therapeutics depends on predictions of clinical activity from pre-clinical data. We have previously described SYNB1618, an engineered bacterial therapeutic (synthetic biotic) for the treatment of Phenylketonuria (PKU), a rare genetic disease that leads to accumulation of plasma phenylalanine (Phe) and severe neurological complications. SYNB1618 consumes Phe in preclinical models, healthy human volunteers, and PKU patients. However, it remains unclear to what extent Phe consumption by SYNB1618 in the gastrointestinal tract lowers plasma Phe levels in PKU patients. Here, we construct a mechanistic model that predicts SYNB1618 function in non-human primates and healthy subjects by combining in vitro simulations and prior knowledge of human physiology. In addition, we extend a model of plasma Phe kinetics in PKU patients, in order to estimate plasma Phe lowering by SYNB1618. This approach provides a framework that can be used more broadly to define the therapeutic potential of synthetic biotics.

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.

Variability and Lability of Ammonia Levels in Healthy Volunteers and Patients With Cirrhosis: Implications for Trial Design and Clinical Practice.

INTRODUCTION: Ammonia levels are used to assess hepatic encephalopathy, but their levels are highly variable in clinical practice. METHODS: We studied factors associated with variation in ammonia values in cirrhotic patients without previous hepatic encephalopathy and healthy volunteers (HVs). RESULTS: Ammonia increased by 12% and 18% at 1 and 2 hour, respectively, after a protein meal in 64 cirrhotic patients (P < 0.001). In 237 HVs, ammonia levels varied significantly between sites (P < 0.0001). New site-specific ammonia upper limits based on HV levels using a strict analysis protocol differed from routinely used values. Correlation between paired fresh samples was high (r = 0.83) but modest between fresh and frozen samples (r = 0.62). DISCUSSION: Sample handling, processing, and protein intake impact ammonia levels across sites.

Genomic and transcriptomic association studies identify 16 novel susceptibility loci for venous thromboembolism.

Venous thromboembolism (VTE) is a significant contributor to morbidity and mortality. To advance our understanding of the biology contributing to VTE, we conducted a genome-wide association study (GWAS) of VTE and a transcriptome-wide association study (TWAS) based on imputed gene expression from whole blood and liver. We meta-analyzed GWAS data from 18 studies for 30 234 VTE cases and 172 122 controls and assessed the association between 12 923 718 genetic variants and VTE. We generated variant prediction scores of gene expression from whole blood and liver tissue and assessed them for association with VTE. Mendelian randomization analyses were conducted for traits genetically associated with novel VTE loci. We identified 34 independent genetic signals for VTE risk from GWAS meta-analysis, of which 14 are newly reported associations. This included 11 newly associated genetic loci (C1orf198, PLEK, OSMR-AS1, NUGGC/SCARA5, GRK5, MPHOSPH9, ARID4A, PLCG2, SMG6, EIF5A, and STX10) of which 6 replicated, and 3 new independent signals in 3 known genes. Further, TWAS identified 5 additional genetic loci with imputed gene expression levels differing between cases and controls in whole blood (SH2B3, SPSB1, RP11-747H7.3, RP4-737E23.2) and in liver (ERAP1). At some GWAS loci, we found suggestive evidence that the VTE association signal for novel and previously known regions colocalized with expression quantitative trait locus signals. Mendelian randomization analyses suggested that blood traits may contribute to the underlying risk of VTE. To conclude, we identified 16 novel susceptibility loci for VTE; for some loci, the association signals are likely mediated through gene expression of nearby genes.

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.

ADP Platelet Hyperreactivity Predicts Cardiovascular Disease in the FHS (Framingham Heart Study).

BACKGROUND: Platelet function is associated with adverse events in patients with cardiovascular disease (CVD). METHODS AND RESULTS: We examined associations of baseline platelet function with incident CVD events in the community-based FHS (Framingham Heart Study). Participants free of prevalent CVD and without recent aspirin treatment with available data in the Framingham Offspring cohort (1991-1995) and Omni cohort (1994-1998) were included. Platelet function was measured with light transmission aggregometry using collagen (1.9 µg/mL), ADP (0.05-15 µmol/L), and epinephrine (0.01-15 µmol/L). We used proportional hazards models to analyze incident outcomes (myocardial infarction/stroke, CVD, and CVD mortality) with respect to platelet measures. The study sample included 2831 participants (average age, 54.3 years; 57% women). During follow-up (median, 20.4 years), we observed 191 composite incident myocardial infarction or stroke events, 432 incident CVD cases, and 117 CVD deaths. Hyperreactivity to ADP and platelet aggregation at ADP concentration of 1.0 µmol/L were significantly associated with incident myocardial infarction/stroke in a multivariable model (hazard ratio, 1.68 [95% confidence interval, 1.13-2.50] [P=0.011] for hyperreactivity across ADP doses; and hazard ratio, 1.16 [95% confidence interval, 1.02-1.33] [P=0.029] for highest quartile of ADP response at 1.0 µmol/L versus others). No association was observed for collagen lag time or any epinephrine measures with incident myocardial infarction or stroke. CONCLUSIONS: Intrinsic hyperreactivity to low-dose ADP in our community-based sample, who were free of CVD and any antiplatelet therapy, is associated with future arterial thrombosis during a 20-year follow-up. These findings reinforce ADP activation inhibition as a critical treatment paradigm and encourage further study of ADP inhibitor-refractive populations.

Familial Hypercholesterolemia and Type 2 Diabetes in the Old Order Amish.

Alleles associated with lower levels of LDL cholesterol (LDL-C) have recently been associated with an increased risk of type 2 diabetes (T2D), highlighting the complex relationship between LDL-C and diabetes. This observation begs the question of whether LDL-C-raising alleles are associated with a decreased risk of T2D. This issue was recently addressed in a large familial hypercholesterolemia (FH) screening study, which reported a lower prevalence of self-reported diabetes in FH subjects than in age-matched relatives without FH. To extend this observation, we tested the association of FH with diabetes status and glycemia in a large Amish population enriched for the FH-associated APOB R3527Q variant that included 640 APOB R3527Q carriers and 4,683 noncarriers. Each copy of the R3527Q T allele was associated with a 74.9 mg/dL increase in LDL-C. There was little difference in T2D prevalence between subjects with (5.2%) and without (4.5%) the R3527Q allele (P = 0.23), and there was no association between R3527Q variant and impaired fasting glucose, fasting glucose or insulin, or oral glucose tolerance test-derived measures. Our data provide no evidence supporting an association between the APOB R3527Q variant and T2D or glycemia and highlight the asymmetry of the LDL-C-T2D relationship and/or the gene/variant-dependent specificity of the LDL-C-T2D association.

Platelet function as a risk factor for venous thromboembolism in the Framingham Heart Study.

INTRODUCTION: The relationship of venous thromboembolism (VTE) with platelet reactivity is unclear. Platelet function plays a key role in arterial thrombosis. Evidence suggests antiplatelet agents also effects in reducing VTE. Our aim is to describe the role of baseline platelet function in development of VTE in the community-based Framingham Heart Study (FHS) cohort. MATERIALS AND METHODS: Participants in the Framingham Offspring cohort fifth examination and Omni cohort first examination were eligible. We used light transmission aggregometry to measure platelet aggregation in response to collagen and a range of ADP and epinephrine doses. The study population consisted of 2831 participants [average age 54.3years; 57% female]. RESULTS AND CONCLUSIONS: During a median follow-up of 20.4years, we observed 138 incident VTE events. In age-, sex- and cohort-adjusted analysis an increase in collagen lag time was associated with increased risk for incident VTE (HR 1.01 [1.00-1.02]; p=0.049). Increased maximal aggregation to low dose epinephrine (1.0µM) was associated with lower VTE risk (HR 0.84 [0.71-0.99]; p=0.042]). However, additional multivariable analyses attenuated the collagen-VTE and epinephrine-VTE associations to trends, primarily due to adjustment for baseline body mass index (BMI), a VTE risk factor and potential modifier of platelet function. Secondary analyses considering varying follow-up periods, cancer incidence and interim aspirin use did not dramatically affect the collagen and epinephrine trends observed. Baseline platelet aggregability was only weakly associated with incident VTE, and in a paradoxical direction, in a community-based population. Other markers of platelet function and hemostasis could prove to be more useful predictors.

Epidemiology of venous thromboembolism in the Framingham Heart Study.

BACKGROUND: Reports of the crude incidence of venous thromboembolism (VTE) in Western countries vary widely. Data regarding risk factors, incidence and recurrence of VTE from deeply-phenotyped community-based cohort studies are needed. OBJECTIVES: To study the incidence, associated mortality, and predisposing factors of VTE in the prospective, longitudinal community-based Framingham Heart Study. PATIENTS/METHODS: The study sample consisted of the Framingham Heart Study Original, Offspring, Third Generation, and Omni cohorts (N=9754). Incidence rates (IR) were standardized to the 2000 US population. Cox proportional hazards regression models were used to study risk factor associations. RESULTS: During 1995-2014 (total follow-up time 104,091 person-years [median 9.8 (range 0-20) years]), 297 incident VTE events were observed. Age-adjusted IR of VTE was 20.3/10,000 (95% CI 17.9-22.6). Of the events 120 (40%) were pulmonary embolism (PE) and 177 (60%) were deep venous thrombosis (DVT); 29% were unprovoked, 40% provoked, and 31% cancer-related. Cancer-related VTE was associated with high mortality at 30days (24.2%), 1year (66.3%), and 5years (75.6%). In multivariable models, age and obesity, but no other traditional cardiovascular risk factors, were significantly associated with VTE (hazard ratio [HR] per 10-year increase in age 1.69, 95% CI 1.48-1.92; HR for obesity (BMI≥30kg/m(2)) 1.88, 95% CI 1.44-2.45). CONCLUSIONS: We provide data on the epidemiology of VTE. VTE is associated with significant mortality, and prognosis after cancer-related VTE is particularly poor. Traditional cardiovascular risk factors beyond age and obesity are not associated with VTE.

Biomarkers for the prediction of venous thromboembolism in the community.

BACKGROUND &OBJECTIVES: Venous and arterial thrombosis share common pathophysiology. Multiple biomarkers reflecting various biological pathways can predict arterial thrombosis. We studied whether this approach could identify persons at risk of first venous thromboembolism (VTE).

Renal Impairment and Prognosis of Patients with Atrial Fibrillation Undergoing Coronary Intervention - The AFCAS Trial.

BACKGROUND: Renal impairment is a well-known risk factor for cardiovascular complications, but the effect of different stages of renal impairment on thrombotic/thromboembolic and bleeding complications in patients with atrial fibrillation (AF) undergoing percutaneous coronary intervention (PCI) remains largely unknown. We sought to evaluate the incidence and clinical impact of four stages of renal impairment in patients with AF undergoing PCI. METHODS: We assessed renal function by estimated glomerular filtration rate (eGFR) and outcomes in 781 AF patients undergoing PCI by using the data from a prospective European multicenter registry. End-points included all-cause mortality, major adverse cardiac and cerebrovascular events (MACCE) and bleeding events at 12 months. RESULTS: A total of 195 (25%) patients had normal renal function (eGFR ≥90 mL/min), 290 (37%) mild renal impairment (eGFR 60-89), 263 (34%) moderate renal impairment (eGFR 30-59) and 33 (4%) severe renal impairment (eGFR <30). Degree of renal impairment remained an independent predictor of mortality and MACCE in an adjusted a Cox regression model. Even patients with mild renal impairment had a higher risk of all-cause mortality (HR 2.25, 95%CI 1.02-4.98, p=0.04) and borderline risk for MACCE (HR 1.56, 95%CI 0.98- 2.50, p=0.06) compared to those with normal renal function. CONCLUSIONS: Renal impairment is common in patients with AF undergoing PCI and even mild renal impairment has an adverse prognostic effect in these patients requiring multiple antithrombotic medications.

CHADS2, CHA2DS2-VASc and HAS-BLED as predictors of outcome in patients with atrial fibrillation undergoing percutaneous coronary intervention.

INTRODUCTION: CHADS2 and CHA2DS2-VASc scores are used to estimate thromboembolic risk in atrial fibrillation (AF). HAS-BLED is recommended for bleeding risk prediction. Their value in predicting the outcome of AF patients after percutaneous coronary intervention (PCI) is unknown. Thus, our aim was to assess whether these simple risk scores are useful in predicting outcome in these patients. MATERIALS AND METHODS: AFCAS is an observational, multicenter, prospective registry including patients (n=929) with AF referred for PCI. Primary study endpoints were 1) all cause mortality; 2) major adverse events (all-cause mortality, myocardial infarction, repeat revascularization, stent thrombosis, transient ischemic attack, stroke or other arterial thromboembolism; MACCE); and 3) bleeding at 12 months follow-up. CHADS2 and CHA2DS2-VASc scores and a modified HAS-BLED (mHAS-BLED) score (omitting labile INR and liver function) were calculated. RESULTS: Patients were distributed as follows: CHADS2 low 29.5%, intermediate 55.2%, high 15.3%; CHA2DS2-VASc low 9.6%, intermediate 46.0%, high 44.5%. A high CHA2DS2-VASc score was predictive of all-cause mortality (p=0.02), whereas CHADS2 was not. High CHA2DS2-VASc score predicted MACCE (HR 2.24, 95%CI 1.21-4.17, p=0.01), as did a high CHADS2 score (HR 1.60, 95%CI 1.05-2.45, p=0.029). Their predictive performance was only modest (C indexes 0.56-0.57). CHADS2 or CHA2DS2-VASc scores were not associated with bleeding. High mHAS-BLED scores (≥3) were not associated with any of the study outcomes. CONCLUSIONS: High CHA2DS2-VASc score was the best predictor of thrombotic outcomes after PCI in a high risk AF population. High mHAS-BLED score was not predictive of bleeding events. More accurate, simple risk scores are needed.