A Comprehensive Study of the Effects by Sequence Truncation within Inverted Terminal Repeats (ITRs) on the Productivity, Genome Packaging, and Potency of AAV Vectors.

One of the primary challenges in working with adeno-associated virus (AAV) lies in the inherent instability of its inverted terminal repeats (ITRs), which play vital roles in AAV replication, encapsidation, and genome integration. ITRs contain a high GC content and palindromic structure, which occasionally results in truncations and mutations during plasmid amplification in bacterial cells. However, there is no thorough study on how these alterations in ITRs impact the ultimate AAV vector characteristics. To close this gap, we designed ITRs with common variations, including a single B, C, or D region deletion at one end, and dual deletions at both ends of the vector genome. These engineered ITR-carrying plasmids were utilized to generate AAV vectors in HEK293 cells. The crude and purified AAV samples were collected and analyzed for yield, capsid DNA-filled percentage, potency, and ITR integrity. The results show that a single deletion had minor impact on AAV productivity, packaging efficiency, and in vivo potency. However, deletions on both ends, except A, showed significant negative effects on the above characteristics. Our work revealed the role of ITR regions, A, B, C, and D for AAV production and DNA replication, and proposes a new strategy for the quality control of ITR-bearing plasmids and final AAV products.

Safety and target engagement of an oral small-molecule sequestrant in adolescents with autism spectrum disorder: an open-label phase 1b/2a trial.

Autism spectrum disorder (ASD) is defined by hallmark behaviors involving reduced communication and social interaction as well as repetitive activities and restricted interests. ASD represents a broad spectrum, from minimally affected individuals to those requiring intense support, with additional manifestations often including anxiety, irritability/aggression and altered sensory processing. Gastrointestinal (GI) issues are also common in ASD, and studies have identified changes in the gut microbiome of individuals with ASD compared to control populations, complementing recent findings of differences in gut-derived metabolites in feces and circulation. However, a role for the GI tract or microbiome in ASD remains controversial. Here we report that an oral GI-restricted adsorbent (AB-2004) that has affinity for small aromatic or phenolic molecules relieves anxiety-like behaviors that are driven by a gut microbial metabolite in mice. Accordingly, a pilot human study was designed and completed to evaluate the safety of AB-2004 in an open-label, single-cohort, multiple-ascending-dose clinical trial that enrolled 30 adolescents with ASD and GI symptoms in New Zealand and Australia. AB-2004 was shown to have good safety and tolerability across all dose levels, and no drug-related serious adverse events were identified. Significant reductions in specific urinary and plasma levels of gut bacterial metabolites were observed between baseline and end of AB-2004 treatment, demonstrating likely target engagement. Furthermore, we observed improvements in multiple exploratory behavioral endpoints, most significantly in post hoc analysis of anxiety and irritability, as well as GI health, after 8 weeks of treatment. These results from an open-label study (trial registration no. ACTRN12618001956291) suggest that targeting gut-derived metabolites with an oral adsorbent is a safe and well-tolerated approach to improving symptoms associated with ASD, thereby emboldening larger placebo-controlled trials.

Plasma and Fecal Metabolite Profiles in Autism Spectrum Disorder.

BACKGROUND: Autism spectrum disorder (ASD) is a neurodevelopmental condition with hallmark behavioral manifestations including impaired social communication and restricted repetitive behavior. In addition, many affected individuals display metabolic imbalances, immune dysregulation, gastrointestinal dysfunction, and altered gut microbiome compositions. METHODS: We sought to better understand nonbehavioral features of ASD by determining molecular signatures in peripheral tissues through mass spectrometry methods (ultrahigh performance liquid chromatography-tandem mass spectrometry) with broad panels of identified metabolites. Herein, we compared the global metabolome of 231 plasma and 97 fecal samples from a large cohort of children with ASD and typically developing control children. RESULTS: Differences in amino acid, lipid, and xenobiotic metabolism distinguished ASD and typically developing samples. Our results implicated oxidative stress and mitochondrial dysfunction, hormone level elevations, lipid profile changes, and altered levels of phenolic microbial metabolites. We also revealed correlations between specific metabolite profiles and clinical behavior scores. Furthermore, a summary of metabolites modestly associated with gastrointestinal dysfunction in ASD is provided, and a pilot study of metabolites that can be transferred via fecal microbial transplant into mice is identified. CONCLUSIONS: These findings support a connection between metabolism, gastrointestinal physiology, and complex behavioral traits and may advance discovery and development of molecular biomarkers for ASD.

Association of Osteopontin, Neopterin, and Myeloperoxidase With Stroke Risk in Patients With Prior Stroke or Transient Ischemic Attacks: Results of an Analysis of 13 Biomarkers From the Stroke Prevention by Aggressive Reduction in Cholesterol Levels Trial.

BACKGROUND AND PURPOSE: Established risk factors do not fully identify patients at risk for recurrent stroke. The SPARCL trial (Stroke Prevention by Aggressive Reduction in Cholesterol Levels) evaluated the effect of atorvastatin on stroke risk in patients with a recent stroke or transient ischemic attack and no known coronary heart disease. This analysis explored the relationships between 13 plasma biomarkers assessed at trial enrollment and the occurrence of outcome strokes. METHODS: We conducted a case-cohort study of 2176 participants; 562 had outcome strokes and 1614 were selected randomly from those without outcome strokes. Time to stroke was evaluated by Cox proportional hazards models. RESULTS: There was no association between time to stroke and lipoprotein-associated phospholipase A2, monocyte chemoattractant protein-1, resistin, matrix metalloproteinase-9, N-terminal fragment of pro-B-type natriuretic peptide, soluble vascular cell adhesion molecule-1, soluble intercellular adhesion molecule-1, or soluble CD40 ligand. In adjusted analyses, osteopontin (hazard ratio per SD change, 1.362; P<0.0001), neopterin (hazard ratio, 1.137; P=0.0107), myeloperoxidase (hazard ratio, 1.177; P=0.0022), and adiponectin (hazard ratio, 1.207; P=0.0013) were independently associated with outcome strokes. After adjustment for the Stroke Prognostic Instrument-II and treatment, osteopontin, neopterin, and myeloperoxidase remained independently associated with outcome strokes. The addition of these 3 biomarkers to Stroke Prognostic Instrument-II increased the area under the receiver operating characteristic curve by 0.023 (P=0.015) and yielded a continuous net reclassification improvement (29.1%; P<0.0001) and an integrated discrimination improvement (42.3%; P<0.0001). CONCLUSIONS: Osteopontin, neopterin, and myeloperoxidase were independently associated with the risk of recurrent stroke and improved risk classification when added to a clinical risk algorithm. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique Identifier: NCT00147602.

Targeting Anti-TGF-ß Therapy to Fibrotic Kidneys with a Dual Specificity Antibody Approach.

Targeted delivery of a therapeutic agent to a site of pathology to ameliorate disease while limiting exposure at undesired tissues is an aspirational treatment scenario. Targeting diseased kidneys for pharmacologic treatment has had limited success. We designed an approach to target an extracellular matrix protein, the fibronectin extra domain A isoform (FnEDA), which is relatively restricted in distribution to sites of tissue injury. In a mouse unilateral ureteral obstruction (UUO) model of renal fibrosis, injury induced significant upregulation of FnEDA in the obstructed kidney. Using dual variable domain Ig (DVD-Ig) technology, we constructed a molecule with a moiety to target FnEDA and a second moiety to neutralize TGF-ß After systemic injection of the bispecific TGF-ß + FnEDA DVD-Ig or an FnEDA mAb, chemiluminescent detection and imaging with whole-body single-photon emission computed tomography (SPECT) revealed significantly higher levels of each molecule in the obstructed kidney than in the nonobstructed kidney, the ipsilateral kidney of sham animals, and other tissues. In comparison, a systemically administered TGF-ß mAb accumulated at lower concentrations in the obstructed kidney and exhibited a more diffuse whole-body distribution. Systemic administration of the bispecific DVD-Ig or the TGF-ß mAb (1-10 mg/kg) but not the FnEDA mAb attenuated the injury-induced collagen deposition detected by immunohistochemistry and elevation in Col1a1, FnEDA, and TIMP1 mRNA expression in the obstructed kidney. Overall, systemic delivery of a bispecific molecule targeting an extracellular matrix protein and delivering a TGF-ß mAb resulted in a relatively focal uptake in the fibrotic kidney and reduced renal fibrosis.

Arabinosylation of recombinant human immunoglobulin-based protein therapeutics.

Protein glycosylation is arguably the paramount post-translational modification on recombinant glycoproteins, and highly cited in the literature for affecting the physiochemical properties and the efficacy of recombinant glycoprotein therapeutics. Glycosylation of human immunoglobulins follows a reasonably well-understood metabolic pathway, which gives rise to a diverse range of asparagine-linked (N-linked), or serine/threonine-linked (O-linked) glycans. In N-linked glycans, fucose levels have been shown to have an inverse relationship with the degree of antibody-dependent cell-mediated cytotoxicity, and high mannose levels have been implicated in potentially increasing immunogenicity and contributing to less favorable pharmacokinetic profiles. Here, we demonstrate a novel approach to potentially reduce the presence of high-mannose species in recombinant human immunoglobulin preparations, as well as facilitate an approximate 100% replacement of fucosylation with arabinosylation in Chinese hamster ovary cell culture through media supplementation with D-arabinose, an uncommonly used mammalian cell culture sugar substrate. The replacement of fucose with arabinose was very effective and practical to implement, since no cell line engineering or cellular adaptation strategies were required. Arabinosylated recombinant IgGs and the accompanying reduction in high mannose glycans, facilitated a reduction in dendritic cell uptake, increased FcγRIIIa signaling, and significantly increased the levels of ADCC. These aforementioned effects were without any adverse changes to various structural or functional attributes of multiple recombinant human antibodies and a bispecific DVD-Ig. Protein arabinosylation represents an expansion of the N-glycan code in mammalian expressed glycoproteins.

Generation and characterization of ABBV642, a dual variable domain immunoglobulin molecule (DVD-Ig) that potently neutralizes VEGF and PDGF-BB and is designed for the treatment of exudative age-related macular degeneration.

Exudative age-related macular degeneration (AMD) is the most common cause of moderate and severe vision loss in developed countries. Intraocular injections of vascular endothelial growth factor (VEGF or VEGF-A)-neutralizing proteins provide substantial benefit, but frequent, long-term injections are needed. In addition, many patients experience initial visual gains that are ultimately lost due to subretinal fibrosis. Preclinical studies and early phase clinical trials suggest that combined suppression of VEGF and platelet-derived growth factor-BB (PDGF-BB) provides better outcomes than suppression of VEGF alone, due to more frequent regression of neovascularization (NV) and suppression of subretinal fibrosis. We generated a dual variable domain immunoglobulin molecule, ABBV642 that specifically and potently binds and neutralizes VEGF and PDGF-BB. ABBV642 has been optimized for treatment of exudative AMD based on the following design characteristics: 1) high affinity binding to all VEGF-A isoforms and both soluble and extracellular matrix (ECM)-associated PDGF-BB; 2) potential for extended residence time in the vitreous cavity to decrease the frequency of intraocular injections; 3) rapid clearance from systemic circulation compared with molecules with wild type Fc region for normal FcRn binding, which may reduce the risk of systemic complications; and 4) low risk of potential effector function. The bispecificity of ABBV642 allows for a single injection of a single therapeutic agent, and thus a more streamlined development and regulatory path compared with combination products. In a mouse model of exudative AMD, ABBV642 was observed to be more effective than aflibercept. ABBV642 has potential to improve efficacy with reduced injection frequency in patients with exudative AMD, thereby reducing the enormous disease burden for patients and society.

Effect of atorvastatin on C-reactive protein and benefits for cardiovascular disease in patients with type 2 diabetes: analyses from the Collaborative Atorvastatin Diabetes Trial.

AIMS/HYPOTHESIS: We investigated whether atorvastatin 10 mg daily lowered C-reactive protein (CRP) and whether the effects of atorvastatin on cardiovascular disease (CVD) varied by achieved levels of CRP and LDL-cholesterol. METHODS: CRP levels were measured at baseline and 1 year after randomisation to atorvastatin in 2,322 patients with type 2 diabetes (40-75 years, 69% males) in a secondary analysis of the Collaborative Atorvastatin Diabetes Study, a randomised placebo-controlled trial. We used Cox regression models to test the effects on subsequent CVD events (n = 147) of CRP and LDL-cholesterol lowering at 1 year. RESULTS: After 1 year, the atorvastatin arm showed a net CRP lowering of 32% (95% CI -40%, -22%) compared with placebo. The CRP response was highly variable, with 45% of those on atorvastatin having no decrease in CRP (median [interquartile range, IQR] per cent change -9.8% [-57%, 115%]). The LDL-cholesterol response was less variable, with a median (IQR) within-person per cent change of -41% (-51%, -31%). Baseline CRP did not predict CVD over 3.8 years of follow-up (HRper SD log 0.89 [95% CI 0.75, 1.06]), whereas baseline LDL-cholesterol predicted CVD (HRper SD 1.21 [95% CI 1.02, 1.44]), as did on-treatment LDL-cholesterol. There was no significant difference in the reduction in CVD by atorvastatin, with above median (HR 0.57) or below median (HR 0.52) change in CRP or change in LDL-cholesterol (HR 0.61 vs 0.50). CONCLUSIONS/INTERPRETATION: CRP was not a strong predictor of CVD. Statin efficacy did not vary with achieved CRP despite considerable variability in CRP response. The use of CRP as an indicator of efficacy of statin therapy on CVD risk in patients with type 2 diabetes is not supported by these data. Trial registration NCT00327418.

Prediction of cardiovascular events in statin-treated stable coronary patients of the treating to new targets randomized controlled trial by lipid and non-lipid biomarkers.

UNLABELLED: Several plasma non-lipid biomarkers have been shown to predict major cardiovascular events (MCVEs) in population studies. Our objective was to investigate the relationship between lipid and non-lipid biomarkers levels achieved during statin therapy and the incidence of MCVEs in patients with stable coronary heart disease (CHD). We conducted a substudy of the TNT (Treating to New Targets) study, which was a randomized trial that compared the efficacy of high (80 mg) versus low (10 mg) dose atorvastatin for the secondary prevention of CHD. Fasting plasma levels of standard lipids and of 18 non-lipid biomarkers were obtained after an 8-week run-in period on atorvastatin 10 mg in 157 patients who experienced MCVEs during the 4.9 years of study follow-up and in 1349 controls. MCVE was defined as CHD death, nonfatal, non-procedure-related myocardial infarction, resuscitated cardiac arrest, and fatal or nonfatal stroke. After adjusting for age, sex and treatment arm, plasma levels of high-density lipoprotein (HDL) cholesterol, triglycerides, high-sensitivity C-reactive protein (hsCRP), insulin, neopterin, N-terminal pro-brain natriuretic peptide (BNP), lipoprotein(a) [Lp(a)], and the soluble receptor for advanced glycation end products (sRAGE) were predictive of recurrent MCVEs (P ≤ 0.02 for each doubling of plasma concentration). However, no significant association was observed between the risk of recurrent MCVEs and plasma levels of low-density lipoprotein cholesterol, adiponectin, cystatin C, lipoprotein-associated phospholipase A2, monocyte chemotactic protein-1, matrix metalloproteinase-9, myeloperoxidase, osteopontin, soluble CD40 ligand, soluble intercellular adhesion molecule-1, or soluble vascular cell adhesion molecule-1. After further adjustment for diabetes, hypertension, smoking, and BMI, the relationship between hsCRP, insulin and MCVE were no longer significant, while the relationship between Lp(a), neopterin, NT-proBNP and sRAGE and MCVE remained statistically significant. In conclusion, in patients with CHD treated with atorvastatin, plasma levels of Lp(a), neopterin, NT-proBNP, and sRAGE are associated with the risk of recurrent MCVEs. TRIAL REGISTRATION: ClinicalTrials.gov NCT00327691.

Total soluble and endogenous secretory receptor for advanced glycation end products as predictive biomarkers of coronary heart disease risk in patients with type 2 diabetes: an analysis from the CARDS trial.

OBJECTIVE: Circulating levels of soluble receptor for advanced glycation end products (sRAGE) likely comprise both a secreted isoform (esRAGE) and wild-type RAGE cleaved from the cell membrane. Both sRAGE and esRAGE have been proposed as biomarkers of cardiovascular disease (CVD), but prospective data are limited. We examined the relationship of sRAGE and esRAGE to incident coronary heart disease (CHD) and stroke in type 2 diabetic patients followed for 3.9 years in a trial of atorvastatin: the Collaborative Atorvastatin Diabetes Study (CARDS). RESEARCH DESIGN AND METHODS: We used a nested case-control design sampling all incident cases of CVD with available plasma and randomly selecting three control subjects, who were free of CVD throughout follow-up, per case. Analysis was by Cox regression with adjustment for treatment allocation and relevant covariates. RESULTS: sRAGE and esRAGE were strongly correlated (ρ = 0.88) and were both higher in those with lower BMI (P < 0.001), higher adiponectin (P < 0.001), lower estimated glomerular filtration rate (P = 0.009), and white ethnicity (P < 0.001). Both sRAGE and esRAGE were associated with incident CHD events, independently of treatment allocation and the above factors; hazard ratio (HR) = 1.74 (95% CI 1.25-2.41; P = 0.002) for a doubling of the sRAGE level; HR = 1.45 (1.11-1.89; P = 0.006) for a doubling of the esRAGE level. There was no significant association with stroke; HR for sRAGE = 0.66 (0.38-1.14). Atorvastatin, 10 mg daily, did not alter sRAGE. CONCLUSIONS: Higher levels of sRAGE and esRAGE are associated with incident CHD but not stroke in type 2 diabetes.

Prediction of cardiovascular events in statin-treated stable coronary patients by lipid and nonlipid biomarkers.

OBJECTIVES: The aim of this study was to investigate the relationship between lipid and nonlipid biomarker levels achieved during statin therapy and the incidence of major cardiovascular events (MCVEs) in patients with stable coronary heart disease (CHD). BACKGROUND: Several plasma nonlipid biomarkers have been shown to predict MCVEs in population studies. METHODS: This is a nested case-control study in the TNT (Treating to New Targets) study population, a randomized trial that compared the efficacy of high- (80 mg) versus low-dose (10 mg) atorvastatin for the secondary prevention of CHD. Fasting plasma levels of standard lipids and of 18 nonlipid biomarkers were obtained after an 8-week run-in period on atorvastatin 10 mg and again 1 year after being randomized to 10 or 80 mg atorvastatin in 507 patients who experienced MCVEs during the 4.9 years of study follow-up and in 1,020 control subjects. An MCVE was defined as CHD death; nonfatal, non-procedure-related myocardial infarction; resuscitated cardiac arrest; or fatal or nonfatal stroke. RESULTS: Low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglycerides were all predictive of recurrent MCVEs (p ≤ 0.009). Concentrations of many of the 18 nonlipid biomarkers were lowered by atorvastatin therapy (independent of dose). However, almost none of the nonlipid biomarker levels, whether measured after the 8-week run-in period or after 1 year of treatment with 10 or 80 mg atorvastatin, were predictive of recurrent MCVEs. CONCLUSIONS: In patients with stable CHD, atorvastatin improved plasma levels of an expanded panel of nonlipid biomarkers. However, independently of atorvastatin dose, the achieved levels of the vast majority of nonlipid biomarkers did not predict MCVEs. (A Study to Determine the Degree of Additional Reduction in CV Risk in Lowering LDL Below Minimum Target Levels [TNT]; NCT00327691).

Effects of multiple doses of the DPP-IV inhibitor PF-734200 on the relationship between GLP-1 and glucose in subjects with type 2 diabetes mellitus.

A randomized, placebo-controlled study evaluated the effects multiple-doses (28 days) dipeptidyl peptidase-IV (DPP-IV) inhibitor PF-734200 on DPP-IV activity, glucose, glucagon-like peptide-1 (GLP-1), glucagon and insulin levels in 72 subjects with type 2 diabetes. The relationship between changes in active GLP-1 and glucose during a meal test appeared non-linear.

Elevated Serum Sorbitol and not Fructose in Type 2 Diabetic Patients.

Reductions in fasting serum fructose or erythrocyte sorbitol have been proposed as markers for early proof of mechanism in clinical development of aldose reductase (AR) inhibitors. However fructose is significantly impacted by meals and evaluation of erythrocyte sorbitol poses technical challenges. To more accurately assess the performance of these markers in biological samples, a gas chromatography-mass spectrometry assay was modified and validated. Serum was collected on three consecutive days from 13 healthy volunteers (HV) and 14 patients with type 2 diabetes mellitus (T2DM), and assayed for sorbitol and fructose using this assay. Serum fructose and sorbitol were relatively constant across the three days. Fasting fructose levels were comparable between the two groups (T2DM: 1.48 +/- 0.49 mg/L; HV: 1.39 +/- 0.38 mg/L, mean +/- standard deviation, P = 0.61), but fasting sorbitol levels were significantly higher in diabetics (T2DM: 0.280 +/- 0.163 mg/L; HV: 0.164 +/- 0.044 mg/L, P = 0.02). Feeding resulted in a 5-6 fold increase in serum fructose levels, but only a 5%-10% increase in sorbitol. Only sorbitol remained significantly elevated pre- and post feeding in T2DM patients relative to HV. These data suggest that serum sorbitol may be a robust proof of mechanism biomarker and facilitate dose selection for clinical development of AR inhibitors.

Pharmacokinetics, pharmacodynamics, tolerability, and safety of a novel sorbitol dehydrogenase inhibitor in healthy participants.

Increased glucose flux through the polyol pathway and the resultant oxidative stress is thought to be a major mechanistic contributor to microvascular diabetic complications. Inhibition of flux through this pathway can be blocked through inhibition of either of 2 enzymes, aldose reductase (AR) or sorbitol dehydrogenase (SDH). This report describes the pharmacokinetics, biomarker pharmacodynamics, and safety of CP-642,931, a potent and specific sorbitol dehydrogenase inhibitor (SDI). CP-642,931 was administered for 7 days to 57 healthy volunteers in doses ranging from 1 to 35 mg daily. After the 35-mg dose, CP-642,931 showed a t((1/2)) of 20.1 hours and t(max) at 0.5 to 1.25 hours. After a 35-mg dose, maximum inhibition of SDH was 91% (on days 1 and 7), and maximum serum sorbitol increase was 152-fold on day 7 compared to control. Five participants discontinued the study due to adverse events, including myalgia, muscle spasm, and muscle fatigue. All symptoms resolved in all but 1 participant, who continued to report intermittent muscle fasciculations upon follow-up. In conclusion, CP-642,931 is a potent and specific SDI that is rapidly absorbed through the oral route and effectively inhibits SDH. However, the drug is not well tolerated due to adverse neuromuscular effects.

Non-linear increase in GLP-1 levels in response to DPP-IV inhibition in healthy adult subjects.

AIM: Dipeptidyl peptidase-IV (DPP-IV) inhibitors represent a new promising therapeutic intervention for the treatment of type 2 diabetes mellitus. The aim of this study was to investigate the effects of DPP-IV inhibition by PF-00734200, a potent competitive DPP-IV inhibitor, on the dynamics of DPP-IV activity and glucagon-like peptide-1 (GLP-1) kinetics in healthy adult subjects. METHODS: This was a prospective randomized, crossover, placebo-controlled, ascending, single, oral dose study conducted at a clinical research centre. Twenty-seven healthy adult subjects were randomized to receive placebo or PF-00734200 with doses ranging from 0.3 to 300 mg (n = 9 per dose group). Pharmacokinetic and pharmacodynamic end points (DPP-IV activity and GLP-1) were measured prior to, and various times after, dosing. RESULTS: PF-00734200 was well tolerated in all subjects. Pharmacokinetics (PK) data indicate that the drug was rapidly absorbed and declined in a biphasic fashion. Mean maximum concentration and area under concentration curve appeared to increase with doses proportionally. DPP-IV inhibition increased with PF-00734200 concentrations, which can be described by an E(max) model with EC50 approximately being 14 ng/ml. DPP-IV inhibition led to greater GLP-1 level accumulation compared with placebo. Plasma GLP-1 levels stimulated by meals were augmented by DPP-IV inhibition. However, the increase in GLP-1 with DPP-IV inhibition was non-linear and maximized at 10 mg, a dose which resulted in about 75% weighted average DPP-IV inhibition over 24 h and a 2.3-fold increase in GLP-1 over placebo. Moreover, even with near complete inhibition of DPP-IV for over 24 h at the highest PF-00734200 dose levels, the GLP-1 levels actually declined during the night compared with postdinner levels. CONCLUSION: DPP-IV inhibition by PF-00734200 resulted in a non-linear increase in plasma GLP-1 level, suggesting GLP-1 levels may be limited by meal stimulus or by production capacity. In addition, GLP-1 level declined even during maximal DPP-IV inhibition, suggesting that there may be additional pathways of GLP-1 elimination other than DPP-IV enzymatic breakdown.

Circulating succinate is elevated in rodent models of hypertension and metabolic disease.

BACKGROUND: Recent evidence suggests that succinate, long known as an intermediate in the citric acid cycle, may also have a role as a signaling molecule through GPR91 and that activation of this receptor results in blood pressure (BP) elevation via the renin-angiotensin system. We sought to test the hypothesis that GPR91 contributes to BP elevation in hypertension. In addition we investigated whether elevated succinate in diabetes could contribute to the increased rate of gluconeogenesis in that condition. METHODS: Circulating succinate concentration was measured using liquid chromatography tandem mass spectrometry in rodent models of hypertension and metabolic disease as well as in human hypertensives and type 2 diabetics in comparison to control subjects. RESULTS: Elevated succinate was detected in spontaneously hypertensive rats (SHR), ob/ob mice, db/db mice, and fa/fa rats in comparison to their non-diseased controls. The changes in concentration are consistent with activation of GPR91. In contrast, neither human hypertensives nor diabetic patients had elevated succinate in comparison to controls. CONCLUSIONS: These findings are consistent with a role of GPR91 signaling in rodent hypertension and diabetes models but not in the analogous human diseases.

Cholesteryl ester transfer protein variants have differential stability but uniform inhibition by torcetrapib.

Cholesteryl ester transfer protein (CETP) is an important modulator of high density lipoprotein cholesterol in humans and thus considered to be a therapeutic target for preventing cardiovascular disease. The gene encoding CETP has been shown to be highly variable, with multiple single nucleotide polymorphisms responsible for altering both its transcription and sequence. Examining nine missense variants of CETP, we found some had significant associations with CETP mass and high density lipoprotein cholesterol levels. Two variants, Pro-373 and Gln-451, appear to be more stable in vivo, an observation mirrored by partial proteolysis studies performed in vitro. Because these naturally occurring variant proteins are potentially present in clinical populations that will be treated with CETP inhibitors, all commonly occurring haplotypes were tested to determine whether the proteins they encode could be inhibited by torcetrapib, a compound currently in clinical trials in combination with atorvastatin. Torcetrapib behaved similarly with all variants, with no significant differences in inhibition.

Serum levels of thiobarbituric acid reactive substances predict cardiovascular events in patients with stable coronary artery disease: a longitudinal analysis of the PREVENT study.

OBJECTIVES: The objective of this study was to test the predictive value of an oxidative stress biomarker in 634 patients from the Prospective Randomized Evaluation of the Vascular Effects of Norvasc Trial (PREVENT). BACKGROUND: Oxidative stress contributes to mechanisms of atherosclerosis and plaque instability. Biomarkers of oxidation, such as malondialdehyde (MDA), may represent independent indicators of risk for patients with stable coronary artery disease (CAD). METHODS: Serum MDA levels were measured as thiobarbituric acid reactive substances (TBARS) in 634 patients with documented CAD using reverse-phase high-performance liquid chromatography and spectrophotometric approaches. RESULTS: During the three-year study, there were 51 major vascular events such as fatal/nonfatal myocardial infarction, 149 hospitalizations for nonfatal vascular events, and 139 patients underwent a major vascular procedure. At baseline, patients with TBARS levels in the highest quartile had a relative risk (RR) of 3.30 (95% confidence interval [CI] 1.47 to 7.42; p = 0.038) for major vascular events, RR of 4.10 (95% CI 2.55 to 6.60; p < 0.0001) for nonfatal vascular events, and RR of 3.84 (95% CI 2.56 to 5.76; p < 0.0001) for major vascular procedures. The effect of TBARS on events and procedures was also seen in a multivariate model adjusted for inflammatory markers (C-reactive protein, soluble intercellular adhesion molecule-1, interleukin-6), and other risk factors (age, low-density lipoprotein, high-density lipoprotein, total cholesterol, triglycerides, body mass index, and blood pressure). This analysis showed an independent effect of TBARS on major vascular events (p = 0.0149), nonfatal vascular events (p < 0.0001), major vascular procedures (p < 0.001), and all vascular events and procedures (p < 0.0001). CONCLUSIONS: Serum levels of TBARS were strongly predictive of cardiovascular events in patients with stable CAD, independently of traditional risk factors and inflammatory markers.