Efficacy and safety of a synthetic biotic for treatment of phenylketonuria: a phase 2 clinical trial.

Despite available treatment options, many patients with phenylketonuria (PKU) cannot achieve target plasma phenylalanine (Phe) levels1. We previously modified Escherichia coli Nissle 1917 to metabolize Phe in the gut after oral administration (SYNB1618) and designed a second strain (SYNB1934) with enhanced activity of phenylalanine ammonia lyase2,3. In a 14-day open-label dose-escalation study (Synpheny-1, NCT04534842 ), we test a primary endpoint of change from baseline in labeled Phe (D5-Phe AUC0-24; D5-Phe area under the curve (AUC) over 24 hours after D5-Phe administration) in plasma after D5-Phe challenge in adult participants with screening Phe of greater than 600 µM. Secondary endpoints were the change from baseline in fasting plasma Phe and the incidence of treatment-emergent adverse events. A total of 20 participants (ten male and ten female) were enrolled and 15 completed the study treatment. Here, we show that both strains lower Phe levels in participants with PKU: D5-Phe AUC0-24 was reduced by 43% from baseline with SYNB1934 and by 34% from baseline with SYNB1618. SYNB1934 led to a decrease in fasting plasma Phe of 40% (95% CI, -52, -24). There were no serious adverse events or infections. Four participants discontinued because of adverse events, and one withdrew during the baseline period. We show that synthetic biotics can metabolize Phe in the gut, lower post-prandial plasma Phe levels and lower fasting plasma Phe in patients with PKU.

Phase I Study of SYNB1891, an Engineered E. coli Nissle Strain Expressing STING Agonist, with and without Atezolizumab in Advanced Malignancies.

PURPOSE: SYNB1891 is a live, modified strain of the probiotic Escherichia coli Nissle 1917 (EcN) engineered to produce cyclic dinucleotides under hypoxia, leading to STimulator of INterferon Genes (STING) activation in phagocytic antigen-presenting cells in tumors and activating complementary innate immune pathways. PATIENTS AND METHODS: This first-in-human study (NCT04167137) enrolled participants with refractory advanced cancers to receive repeat intratumoral injections of SYNB1891 either alone or in combination with atezolizumab, with the primary objective of evaluating the safety and tolerability of both regimens. RESULTS: Twenty-four participants received monotherapy across six cohorts, and 8 participants received combination therapy in two cohorts. Five cytokine release syndrome events occurred with monotherapy, including one that met the criteria for dose-limiting toxicity at the highest dose; no other SYNB1891-related serious adverse events occurred, and no SYNB1891-related infections were observed. SYNB1891 was not detected in the blood at 6 or 24 hours after the first intratumoral dose or in tumor tissue 7 days following the first dose. Treatment with SYNB1891 resulted in activation of the STING pathway and target engagement as assessed by upregulation of IFN-stimulated genes, chemokines/cytokines, and T-cell response genes in core biopsies obtained predose and 7 days following the third weekly dose. In addition, a dose-related increase in serum cytokines was observed, as well as stable disease in 4 participants refractory to prior PD-1/L1 antibodies. CONCLUSIONS: Repeat intratumoral injection of SYNB1891 as monotherapy and in combination with atezolizumab was safe and well tolerated, and evidence of STING pathway target engagement was observed.

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.

Development of synthetic biotics as treatment for human diseases.

Advances in synthetic biology have allowed the generation of strains of bacteria that are genetically altered to have specific therapeutic benefits. These synthetic biotics, also widely referred to as engineered living therapeutics, have tremendous potential as a new therapeutic modality, and several have advanced into the clinic and human testing. This review outlines some of the unique attributes of synthetic biotics as well as some of the challenges in their development as prescription products. Regulatory considerations are discussed, and a case study of a program that has advanced into Phase 2 testing is provided: SYNB1618 for the treatment of PKU.

COVID-19 Outbreak Response for an Emergency Department Using In Situ Simulation.

In January 2020, the WHO declared COVID-19 an epidemic in China and the possibility of evolving into a pandemic. During early phases, most emergency departments did not have contingency plans for an outbreak of this scale and therefore necessitating adequate preparation. This study aims to describe the utility of in situ simulation in identifying system errors and latent safety hazards in response to preparation for the expected COVID-19 surge. We also aim to describe the corrective measures taken to improve our outbreak response locally. We hope that others may be able to use this information as foresight in preparing their own departments for this outbreak. The emergency department and anesthesiology department of Galway University Hospital conducted a series of multidisciplinary, in situ simulations to rapidly identify operational errors and latent safety hazards in response to this outbreak. Each simulation involved an interdisciplinary response to a suspected/COVID-19 patient. The cases were used as a training opportunity for staff and ultimately a platform to expeditiously implement system changes in response to deficits identified during the simulations. Conclusively, with the complexities and intricate structure of every emergency department, we understood that preparation for an outbreak requires evaluation of the current system before implementing any changes. It is not a "one size fits all" concept. Therefore, conducting in situ simulations and the use of foresight, is pivotal as it could prevent loss of resources and time in preparing for an outbreak.

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.

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.

Translational Development of Microbiome-Based Therapeutics: Kinetics of E. coli Nissle and Engineered Strains in Humans and Nonhuman Primates.

Understanding the pharmacology of microbiome-based therapeutics is required to support the development of new medicines. Strains of E. coli Nissle (EcN) were genetically modified and administered to cynomolgus monkeys at doses of 1 × 109 and 1 × 1012 colony-forming units (CFU)/day for 28 days. A clinical study to evaluate the exposure and clearance of EcN in healthy volunteers was also performed. Healthy subjects received oral doses of EcN, 2.5 to 25 × 109 CFU 3 times daily for 28 days or a single day. In cynomolgus monkeys, replicating strains yielded higher fecal concentrations than nonreplicating strains and persisted for longer following cessation of dosing. In the clinical study, all subjects cleared EcN following cessation of dosing with median clearance of 1 week. Quantitative methodology can be applied to microbiome-based therapeutics, and similar kinetics and clearance were observed for EcN in cynomolgus monkeys and humans.

The effects of caffeinated and decaffeinated coffee on sex hormone-binding globulin and endogenous sex hormone levels: a randomized controlled trial.

BACKGROUND: Findings from observational studies suggest that sex hormone-binding globulin (SHBG) and endogenous sex hormones may be mediators of the putative relation between coffee consumption and lower risk of type 2 diabetes. The objective of this study was to evaluate the effects of caffeinated and decaffeinated coffee on SHBG and sex hormone levels. FINDINGS: After a two-week run-in phase with caffeine abstention, we conducted an 8-week parallel-arm randomized controlled trial. Healthy adults (n = 42) were recruited from the Boston community who were regular coffee consumers, nonsmokers, and overweight. Participants were randomized to five 6-ounce cups of caffeinated or decaffeinated instant coffee or water (control group) per day consumed with each meal, mid-morning, and mid-afternoon. The main outcome measures were SHBG and sex hormones [i.e., testosterone, estradiol, dehydroepiandrosterone sulfate]. No significant differences were found between treatment groups for any of the studied outcomes at week 8. At 4 weeks, decaffeinated coffee was associated with a borderline significant increase in SHBG in women, but not in men. At week 4, we also observed several differences in hormone concentrations between the treatment groups. Among men, consumption of caffeinated coffee increased total testosterone and decreased total and free estradiol. Among women, decaffeinated coffee decreased total and free testosterone and caffeinated coffee decreased total testosterone. CONCLUSIONS: Our data do not indicate a consistent effect of caffeinated coffee consumption on SHBG in men or women, however results should be interpreted with caution given the small sample size. This is the first randomized trial investigating the effects of caffeinated and decaffeinated coffee on SHBG and sex hormones and our findings necessitate further examination in a larger intervention trial.

Short-term walnut consumption increases circulating total adiponectin and apolipoprotein A concentrations, but does not affect markers of inflammation or vascular injury in obese humans with the metabolic syndrome: data from a double-blinded, randomized, placebo-controlled study.

Long-term consumption of walnuts is associated with lower cardiovascular disease risk in epidemiological studies, possibly through improvements in lipid profile and endothelial function. It remains to be elucidated how soon after initiation of walnut consumption beneficial effects on lipid profile and biomarkers of inflammation or vascular injury can be observed. Fifteen obese subjects (9 men and 6 women; age, 58 ± 2.5 years; body mass index, 36.6 ± 1.7 kg/m(2)) with the metabolic syndrome participated as inpatients in a randomized, double-blinded, placebo-controlled crossover study involving short-term placebo or walnut-enriched diet (48 g/d for 4 days). Apolipoproteins and markers of inflammation and vascular injury were measured before and after consumption of the experimental diets. Consumption of walnuts was associated with a statistically significant increase in serum apolipoprotein A concentrations (P = .03), but did not affect circulating levels of fetuin A, resistin, C-reactive protein, serum amyloid A, soluble intercellular adhesion molecules 1 and 3, soluble vascular cell adhesion protein 1, interleukins 6 and 8, tumor necrosis factor α, E-selectin, P-selectin, and thrombomodulin. Four days of walnut consumption (48 g/d) leads to mild increases in apolipoprotein A concentrations, changes that may precede and lead to the beneficial effects of walnuts on lipid profile in obese subjects with the metabolic syndrome.

Effects of caffeinated and decaffeinated coffee on biological risk factors for type 2 diabetes: a randomized controlled trial.

BACKGROUND: Coffee consumption has been associated with a lower risk of type 2 diabetes in prospective cohort studies, but the underlying mechanisms remain unclear. The aim of this study was to evaluate the effects of regular and decaffeinated coffee on biological risk factors for type 2 diabetes. METHODS: Randomized parallel-arm intervention conducted in 45 healthy overweight volunteers who were nonsmokers and regular coffee consumers. Participants were assigned to consumption of 5 cups (177 mL each) per day of instant caffeinated coffee, decaffeinated coffee, or no coffee (i.e., water) for 8 weeks. RESULTS: Average age was 40 years and body mass index was 29.5 kg/m2. Compared with consuming no coffee, consumption of caffeinated coffee increased adiponectin (difference in change from baseline 1.4 µg/mL; 95% CI: 0.2, 2.7) and interleukin-6 (difference: 60%; 95% CI: 8, 138) concentrations and consumption of decaffeinated coffee decreased fetuin-A concentrations (difference: -20%; 95% CI: -35, -1). For measures of glucose tolerance, insulin sensitivity, and insulin secretion, no significant differences were found between treatment groups. CONCLUSIONS: Although no changes in glycemia and/or insulin sensitivity were observed after 8 weeks of coffee consumption, improvements in adipocyte and liver function as indicated by changes in adiponectin and fetuin-A concentrations may contribute to beneficial metabolic effects of long-term coffee consumption. TRIAL REGISTRATION: clinicaltrials.gov NCT00305097.

Leptin administration to overweight and obese subjects for 6 months increases free leptin concentrations but does not alter circulating hormones of the thyroid and IGF axes during weight loss induced by a mild hypocaloric diet.

OBJECTIVE: Short-term energy deprivation reduces leptin concentrations and alters the levels of circulating hormones of the hypothalamic-pituitary-peripheral axis in lean subjects. Whether the reduction in leptin concentration during long-term weight loss in obese individuals is linked to the same neuroendocrine changes seen in lean, leptin-sensitive subjects remains to be fully clarified. METHODS: In this study, 24 overweight and obese adults (16 women and eight men; body mass index (BMI): 27.5-38.0 kg/m(2)) were prescribed a hypocaloric diet (-500 kcal/day) and were randomized to receive recombinant methionyl leptin (n=18, metreleptin, 10 mg/day self-injected s.c.) or placebo (n=6, same volume and time as metreleptin) for 6 months. RESULTS: Metreleptin administration did not affect weight loss beyond that induced by hypocaloric diet alone (P for interaction=0.341) but increased the serum concentrations of total leptin by six- to eight-fold (P<0.001) and led to the generation of anti-leptin antibodies. Despite free leptin concentration (P for interaction=0.041) increasing from 9±1 ng/ml at baseline to 43±15 and 36±12 ng/ml at 3 and 6 months, respectively, changes in circulating hormones of the thyroid and IGF axes at 3 and 6 months were not significantly different in the placebo- and metreleptin-treated groups. CONCLUSIONS: Leptin does not likely mediate changes in neuroendocrine function in response to weight loss induced by a mild hypocaloric diet in overweight and obese subjects.

Efficacy of metreleptin in obese patients with type 2 diabetes: cellular and molecular pathways underlying leptin tolerance.

OBJECTIVE: Metreleptin has been efficacious in improving metabolic control in patients with lipodystrophy, but its efficacy has not been tested in obese patients with type 2 diabetes. RESEARCH DESIGN AND METHODS: We studied the role of leptin in regulating the endocrine adaptation to long-term caloric deprivation and weight loss in obese diabetic subjects over 16 weeks in the context of a double-blinded, placebo-controlled, randomized trial. We then performed detailed interventional and mechanistic signaling studies in humans in vivo, ex vivo, and in vitro. RESULTS: In obese patients with diabetes, metreleptin administration for 16 weeks did not alter body weight or circulating inflammatory markers but reduced HbA(1c) marginally (8.01 ± 0.93-7.96 ± 1.12, P = 0.03). Total leptin, leptin-binding protein, and antileptin antibody levels increased, limiting free leptin availability and resulting in circulating free leptin levels of ∼50 ng/mL. Consistent with clinical observations, all metreleptin signaling pathways studied in human adipose tissue and peripheral blood mononuclear cells were saturable at ∼50 ng/mL, with no major differences in timing or magnitude of leptin-activated STAT3 phosphorylation in tissues from male versus female or obese versus lean humans in vivo, ex vivo, or in vitro. We also observed for the first time that endoplasmic reticulum (ER) stress in human primary adipocytes inhibits leptin signaling. CONCLUSIONS: In obese patients with diabetes, metreleptin administration did not alter body weight or circulating inflammatory markers but reduced HbA(1c) marginally. ER stress and the saturable nature of leptin signaling pathways play a key role in the development of leptin tolerance in obese patients with diabetes.

Effect of iron overload on glucose metabolism in patients with hereditary hemochromatosis.

Diabetes mellitus (DM) affects 30% to 60% of patients with hereditary hemochromatosis (HH). The underlying pathophysiology of DM in patients with hemochromatosis has not been fully elucidated. We studied both insulin secretion and insulin sensitivity in a cohort of patients with HH. We studied glucose metabolism in 53 newly diagnosed HH patients using a standard 75-g oral glucose tolerance test. Basal and stimulated insulin sensitivities were calculated using the quantitative insulin sensitivity check index and oral glucose insulin sensitivity index, respectively. beta-Cell function was assessed using C-peptide concentrations during the oral glucose tolerance test after adjusting for ambient insulin sensitivity. Twenty healthy subjects served as the control group. Fifteen subjects (28%) with HH had abnormal glucose tolerance (AGT). Seven (13%) had DM, and 8 (15%) had impaired glucose tolerance. As well as higher fasting glucose and glycated hemoglobin, those with AGT had a higher fasting insulin and C-peptide levels compared with those with normal glucose tolerance (NGT) (all Ps < .05). Insulin sensitivity measurements showed that the subjects in HH group with AGT were more insulin resistant than the subjects with NGT and controls subjects (P < .05). No significant changes were observed between the groups with NGT and AGT regarding hepatic insulin extraction and both indices related to insulin release in subjects with HH. Our cohort of patients with hemochromatosis and AGT had features similar to typical type 2 DM patients. These findings challenge the traditional view that DM in hemochromatosis is due primarily to iron-induced beta-cell failure.

Walnut consumption increases satiation but has no effect on insulin resistance or the metabolic profile over a 4-day period.

Obesity and diabetes have been associated with increased consumption of highly processed foods, and reduced consumption of whole grains and nuts. It has been proposed, mainly on the basis of observational studies, that nuts may provide superior satiation, may lead to reduced calorie consumption, and may decrease the risk of type 2 diabetes; but evidence from randomized, interventional studies is lacking. A total of 20 men and women with the metabolic syndrome participated in a randomized, double-blind, crossover study of walnut consumption. Subjects had two 4-day admissions to the clinical research center where they were fed an isocaloric diet. In addition, they consumed shakes for breakfast containing either walnuts or placebo (shakes were standardized for calories, carbohydrate, and fat content). Appetite, insulin resistance, and metabolic parameters were measured. We found an increased level of satiety (overall P value = 0.0079) and sense of fullness (P = 0.05) in prelunch questionnaires following the walnut breakfast as compared to the placebo breakfast, with the walnut effect achieving significance on day 3 and 4 (P = 0.02 and P = 0.03). We did not find any change in resting energy expenditure, hormones known to mediate satiety, or insulin resistance when comparing the walnut vs. placebo diet. Walnut consumption over 4 days increased satiety by day 3. Long-term studies are needed to confirm the physiologic role of walnuts, the duration of time needed for these effects to occur, and to elucidate the underlying mechanisms.

Regular consumption of nuts is associated with a lower risk of cardiovascular disease in women with type 2 diabetes.

Higher nut consumption has been associated with lower risk of coronary heart disease (CHD) events in several epidemiologic studies. The study examined the association between intake of nuts and incident cardiovascular disease (CVD) in a cohort of women with type 2 diabetes. For the primary analysis, there were 6309 women with type 2 diabetes who completed a validated FFQ every 2-4 y between 1980 and 2002 and were without CVD or cancer at study entry. Major CVD events included incident myocardial infarction (MI), revascularization, and stroke. During 54,656 person-years of follow-up, there were 452 CHD events (including MI and revascularization) and 182 incident stroke cases. Frequent nut and peanut butter consumption was inversely associated with total CVD risk in age-adjusted analyses. After adjustment for conventional CVD risk factors, consumption of at least 5 servings/wk of nuts or peanut butter [serving size, 28 g (1 ounce) for nuts and 16 g (1 tablespoon) for peanut butter] was significantly associated with a lower risk of CVD (relative risk = 0.56; 95% CI: 0.36-0.89). Furthermore, when we evaluated plasma lipid and inflammatory biomarkers, we observed that increasing nut consumption was significantly associated with a more favorable plasma lipid profile, including lower LDL cholesterol, non-HDL cholesterol, total cholesterol, and apolipoprotein-B-100 concentrations. However, we did not observe significant associations for HDL cholesterol or inflammatory markers. These data suggest that frequent nut and peanut butter consumption is associated with a significantly lower CVD risk in women with type 2 diabetes.

Effect of valsartan on left ventricular anatomy and systolic function and aortic elasticity.

The objective of the study was to examine the effect of a 6-month daily treatment with 160 mg valsartan, an angiotensin II receptor blocker, on the left ventricular systolic function and aortic elasticity of patients with type 2 diabetes mellitus (T2DM) and healthy subjects. This was a prospective, randomized, double-blind, placebo-controlled crossover study. Thirteen healthy control subjects and 11 patients with T2DM were enrolled in the study. Eight control subjects and 4 T2DM patients completed the study. Cardiovascular magnetic resonance was used to evaluate the effect of valsartan on the left ventricular function and aortic elasticity. At baseline, T2DM patients had increased left ventricular mass (P = .006) when compared with the healthy controls. In the T2DM patients, treatment with valsartan, in comparison with receiving placebo, resulted in a reduction of aortic radius (P = .026) and wall thickness (P = .032) of the ascending aorta. In the abdominal aorta, valsartan treatment, when compared with placebo treatment, reduced the arterial compliance (P = .014) in the T2DM patients. Valsartan treatment for 6 months decreased the diameter and wall thickness of the ascending aorta in patients with T2DM, but may decrease AC of the abdominal aorta.

Phobic anxiety is associated with higher serum concentrations of adipokines and cytokines in women with diabetes.

OBJECTIVE: Phobic anxiety has been associated with increased risk of cardiovascular disease (CVD), but the underlying mechanisms are poorly understood. We aimed to determine whether associations of phobic anxiety with several known markers of CVD might be contributors. RESEARCH DESIGN AND METHODS: We used a 16-point validated index (Crown-Crisp) measured in 1988 to categorize 984 women with type 2 diabetes from the Nurses' Health Study as having low, moderate, or high phobic anxiety. Groups were then compared for differences in adipokines (adiponectin and leptin), inflammatory markers (C-reactive protein and tumor necrosis factor [TNF]-alpha receptor II), and markers of endothelial function (sE-selectin, soluble intercellular adhesion molecule [sICAM]-1) measured on blood samples provided between 1989 and 1990. RESULTS: Higher levels of phobic anxiety were associated with higher BMI and lower education. Higher levels of phobic anxiety were also associated with higher leptin and soluble TNF-alpha receptor II in both crude analyses and after adjustment for potential confounders. sICAM and sE-selectin were higher in the highest tertile compared with the middle tertile, but there was no significant trend across tertiles. We found no association between phobic anxiety and adiponectin. CONCLUSIONS: High levels of phobic anxiety are associated with increased levels of leptin and inflammatory markers, which may in part explain the previously observed relationship between anxiety and other psychosocial disorders with CVD.