Aptamer BC007 for neutralization of pathogenic autoantibodies directed against G-protein coupled receptors: A vision of future treatment of patients with cardiomyopathies and positivity for those autoantibodies.

Cardiomyopathies such as idiopathic dilated cardiomyopathy (DCM), Chagas' cardiomyopathy and Peripartum cardiomyopathy present with autoantibodies against G-protein coupled receptors (GPCR-AABs) that agonistically activate their receptors. For the treatment of "agonistic autoantibody diseases" and in particular DCM, the removal of the GPCR-AABs by immunoadsorption (IA) has been studied with convincing patient benefit. To overcome cost and logistics problems of IA, the application of the aptamer BC007 for in vivo neutralization of GPCR-AABs could help. We demonstrate here, that the aptamer neutralized, in vitro, the presently known cardiovascular-pathogenic GPCR-AABs. In spontaneously hypertensive rats, the aptamer demonstrated its GPCR-AAB neutralizing potency in vivo. In the serum of DCM patients, the same GPCR-AAB reduction was achieved when patients were either immunoadsorbed or patient's serum was ex vivo treated with the aptamer. In our view, aptamer BC007 treatment in GPCR-AAB-positive patients would have a comparable benefit as that seen after IA. Not knowing all that interfering with our idea of aptamer-dependent neutralization of GPCR-AABs, the first preliminary steps have been taken for bringing the idea closer to patients.

IsoQC (QPCTL) knock-out mice suggest differential substrate conversion by glutaminyl cyclase isoenzymes.

Secretory peptides and proteins are frequently modified by pyroglutamic acid (pE, pGlu) at their N-terminus. This modification is catalyzed by the glutaminyl cyclases QC and isoQC. Here, we decipher the roles of the isoenzymes by characterization of IsoQC-/- mice. These mice show a significant reduction of glutaminyl cyclase activity in brain and peripheral tissue, suggesting ubiquitous expression of the isoQC enzyme. An assay of substrate conversion in vivo reveals impaired generation of the pGlu-modified C-C chemokine ligand 2 (CCL2, MCP-1) in isoQC-/- mice. The pGlu-formation was also impaired in primary neurons, which express significant levels of QC. Interestingly, however, the formation of the neuropeptide hormone thyrotropin-releasing hormone (TRH), assessed by immunohistochemistry and hormonal analysis of hypothalamic-pituitary-thyroid axis, was not affected in isoQC-/-, which contrasts to QC-/-. Thus, the results reveal differential functions of isoQC and QC in the formation of the pGlu-peptides CCL2 and TRH. Substrates requiring extensive prohormone processing in secretory granules, such as TRH, are primarily converted by QC. In contrast, protein substrates such as CCL2 appear to be primarily converted by isoQC. The results provide a new example, how subtle differences in subcellular localization of enzymes and substrate precursor maturation might influence pGlu-product formation.

Neutralization of pathogenic beta1-receptor autoantibodies by aptamers in vivo: the first successful proof of principle in spontaneously hypertensive rats.

Autoantibodies (AABs) against the second extracellular loop of the beta1-receptor (beta1(II)-AABs) are found as a pathogenic driver in patients with idiopathic dilated cardiomyopathy, Chagas cardiomyopathy, peripartum cardiomyopathy, and myocarditis, and have been increasingly seen as a treatment target. We recently identified an aptamer (single short DNA strand) that specifically binds and neutralizes beta1(II)-AABs. Via application of this aptamer, a new treatment strategy for diseases associated with the cardio-pathogenic beta1(II)-AABs could be developed. Spontaneously hypertensive rats (SHR) positive for beta1(II)-AABs were treated five times at weekly intervals (bolus application of 2 mg/kg body weight followed by an infusion of the same amount over 20 min). SHR responded to aptamer treatment with a strong reduction in the cardio-pathogenic beta1(II)-AABs. The AABs did not substantially return within the study period. No signs for aptamer toxicity were observed by visual examination of the heart, liver, and kidney, or by measurement of plasma CK, ALT, and creatinine. The aptamer's potential for beta1(II)-AAB neutralization and consequently for cardiomyopathy treatment has been shown for the first time in vivo.

The first aptamer-apheresis column specifically for clearing blood of ß1-receptor autoantibodies.

BACKGROUND: Application of immunoapheresis to eliminate pathogenic autoantibodies targeting the second extracellular loop of the ß1-receptor (ß1-AABs) is currently investigated in patients with cardiomyopathy. Aptamers (single short DNA or RNA strands) are a new class of molecules that bind to a specific target molecule. This property qualifies aptamers for potential use in the apheresis technique. We recently identified an aptamer that specifically binds to ß1-AABs, so in the present study we tested whether this aptamer could be used as a binder to prepare an apheresis column suitable for clearing ß1-AABs from rat's blood. METHODS AND RESULTS: An apheresis column was designed containing the ß1-AAB-targeting-aptamer coupled to sepharose. As tested in vitro, this column (1) binds ß1-AABs highly specifically without marked interference with common IgGs, (2) has a capacity for clearing of approximately 1L of ß1-AAB-positive serum and (3) can be completely regenerated for subsequent use. Using the column for extracorporeal apheresis of spontaneously hypertensive rats (SHR) positive for both ß1-AABs and muscarinic 2-receptor autoantibodies (M2-AABs), only ß1-AABs were removed. In a follow-up of 9 weeks, recurrence of ß1-AABs in the blood of SHR could not be detected. CONCLUSIONS: For the first time, a newly designed apheresis column with a ß1-AAB specific aptamer as a binder was successfully used to eliminate ß1-AABs from SHR blood.

Glycaemic variability and pancreatic ß-cell dysfunction.

The importance of glycaemic variability (GV) as a factor in the pathophysiology of cellular dysfunction and late diabetes complications is currently a matter of debate. However, there is mounting evidence from in vivo and in vitro studies that GV has adverse effects on the cascade of physiological processes that result in chronic ß-cell dysfunctions. Glucose fluctuations more than sustained chronic hyperglycaemia can induce excessive formation of reactive oxygen (ROS) and reactive nitrogen species (RNS), ultimately leading to apoptosis related to oxidative stress. The insulin-secreting ß-cells are particularly susceptible to damage imposed by oxidative stress. Evidence from experiments, using isolated pancreatic islets or ß-cell lines, has linked intermittent high glucose, which mimicks GV under diabetic conditions, to significant impairment of ß-cell function. Several clinical studies reported a close association between GV and ß-cell dysfunction, although the deleterious effects are difficult to demonstrate. Notwithstanding, early therapeutic interventions in patients with type 1 as well as type 2 diabetes, using different strategies of optimising glycaemic control, have shown that favourable outcomes on recovery and maintenance of ß-cell function correlated with reduction of GV. The purpose of the present review is to discuss the detrimental effects of GV and associations with ß-cell function as well as upcoming therapeutic strategies directed towards minimising glucose excursions, improving ß-cell recovery and preventing progressive ß-cell loss. Measuring GV has importance for management of diabetes, because it is the only one component of the dysglycaemia that reflects the degree of dysregulation of glucose homeostasis.

The isoenzyme of glutaminyl cyclase is an important regulator of monocyte infiltration under inflammatory conditions.

Acute and chronic inflammatory disorders are characterized by detrimental cytokine and chemokine expression. Frequently, the chemotactic activity of cytokines depends on a modified N-terminus of the polypeptide. Among those, the N-terminus of monocyte chemoattractant protein 1 (CCL2 and MCP-1) is modified to a pyroglutamate (pE-) residue protecting against degradation in vivo. Here, we show that the N-terminal pE-formation depends on glutaminyl cyclase activity. The pE-residue increases stability against N-terminal degradation by aminopeptidases and improves receptor activation and signal transduction in vitro. Genetic ablation of the glutaminyl cyclase iso-enzymes QC (QPCT) or isoQC (QPCTL) revealed a major role of isoQC for pE(1) -CCL2 formation and monocyte infiltration. Consistently, administration of QC-inhibitors in inflammatory models, such as thioglycollate-induced peritonitis reduced monocyte infiltration. The pharmacologic efficacy of QC/isoQC-inhibition was assessed in accelerated atherosclerosis in ApoE3*Leiden mice, showing attenuated atherosclerotic pathology following chronic oral treatment. Current strategies targeting CCL2 are mainly based on antibodies or spiegelmers. The application of small, orally available inhibitors of glutaminyl cyclases represents an alternative therapeutic strategy to treat CCL2-driven disorders such as atherosclerosis/restenosis and fibrosis.

Glutaminyl cyclase knock-out mice exhibit slight hypothyroidism but no hypogonadism: implications for enzyme function and drug development.

Glutaminyl cyclases (QCs) catalyze the formation of pyroglutamate (pGlu) residues at the N terminus of peptides and proteins. Hypothalamic pGlu hormones, such as thyrotropin-releasing hormone and gonadotropin-releasing hormone are essential for regulation of metabolism and fertility in the hypothalamic pituitary thyroid and gonadal axes, respectively. Here, we analyzed the consequences of constitutive genetic QC ablation on endocrine functions and on the behavior of adult mice. Adult homozygous QC knock-out mice are fertile and behave indistinguishably from wild type mice in tests of motor function, cognition, general activity, and ingestion behavior. The QC knock-out results in a dramatic drop of enzyme activity in the brain, especially in hypothalamus and in plasma. Other peripheral organs like liver and spleen still contain QC activity, which is most likely caused by its homolog isoQC. The serum gonadotropin-releasing hormone, TSH, and testosterone concentrations were not changed by QC depletion. The serum thyroxine was decreased by 24% in homozygous QC knock-out animals, suggesting a mild hypothyroidism. QC knock-out mice were indistinguishable from wild type with regard to blood glucose and glucose tolerance, thus differing from reports of thyrotropin-releasing hormone knock-out mice significantly. The results suggest a significant formation of the hypothalamic pGlu hormones by alternative mechanisms, like spontaneous cyclization or conversion by isoQC. The different effects of QC depletion on the hypothalamic pituitary thyroid and gonadal axes might indicate slightly different modes of substrate conversion of both enzymes. The absence of significant abnormalities in QC knock-out mice suggests the presence of a therapeutic window for suppression of QC activity in current drug development.

DPP-4 inhibition increases GIP and decreases GLP-1 incretin effects during intravenous glucose tolerance test in Wistar rats.

GIP metabolite [GIP (3-42)] and GLP-1 metabolite [GLP-1 (9-36) amide] have been reported to differ with regard to biological actions. Systemic DPP-4 inhibition can therefore reveal different actions of GIP and GLP-1. In catheter wearing Wistar rats, insulinotropic effects of equipotent doses of GIP (2.0 nmol/kg) and GLP-1 (7-36) amide (4.0 nmol/kg) and vehicle were tested in the absence/presence of DPP-4 inhibition. Blood glucose and insulin were frequently sampled. DPP-4 inhibitor was given at -20 min, the incretin at -5 min and the intravenous glucose tolerance test (0.4 g glucose/kg) commenced at 0 min. G-AUC and I-AUC, insulinogenic index and glucose efflux, were calculated from glucose and insulin curves. Systemic DPP-4 inhibition potentiated the acute GIP incretin effects: I-AUC (115±34 vs. 153±39 ng·min/ml), increased the insulinogenic index (0.74±0.24 vs. 0.99±0.26 ng/mmol), and improved glucose efflux (19.8±3.1 vs. 20.5±5.0 min⁻¹). The GLP-1 incretin effects were diminished: I-AUC (124±18 vs. 106±38 ng·min/ml), the insulinogenic index was decreased (0.70±0.18 vs. 0.50±0.19 ng/mmol), and glucose efflux declined (14.9±3.1 vs. 11.1±3.7 min⁻¹). GLP-1 and GIP differ remarkably in their glucoregulatory actions in healthy rats when DPP-4 is inhibited. These previously unrecognized actions of DPP-4 inhibitors could have implications for future use in humans.

Glycemic variability correlates strongly with postprandial beta-cell dysfunction in a segment of type 2 diabetic patients using oral hypoglycemic agents.

OBJECTIVE: Glucose fluctuations trigger activation of oxidative stress, a main mechanism leading to secondary diabetes complications. We evaluated the relationship between glycemic variability and beta-cell dysfunction. RESEARCH DESIGN AND METHODS: We conducted a cross-sectional study in 59 patients with type 2 diabetes (aged 64.2 +/- 8.6 years, A1C 6.5 +/- 1.0%, and BMI 29.8 +/- 3.8 kg/m(2)[mean +/- SD]) using either oral hypoglycemic agents (OHAs) (n = 34) or diet alone (nonusers). As a measure of glycemic variability, the mean amplitude of glycemic excursions (MAGE) was computed from continuous glucose monitoring data recorded over 3 consecutive days. The relationships between MAGE, beta-cell function, and clinical parameters were assessed by including postprandial beta-cell function (PBCF) and basal beta-cell function (BBCF) obtained by a model-based method from plasma C-peptide and plasma glucose during a mixed-meal test as well as homeostasis model assessment of insulin sensitivity, clinical factors, carbohydrate intake, and type of OHA. RESULTS: MAGE was nonlinearly correlated with PBCF (r = 0.54, P < 0.001) and with BBCF (r = 0.31, P = 0.025) in OHA users but failed to correlate with these parameters in nonusers (PBCF P = 0.21 and BBCF P = 0.07). The stepwise multiple regression analysis demonstrated that PBCF and OHA combination treatment were independent contributors to MAGE (R(2) = 0.50, P < 0.010), whereas insulin sensitivity, carbohydrate intake, and nonglycemic parameters failed to contribute. CONCLUSIONS: PBCF appears to be an important target to reduce glucose fluctuations in OHA-treated type 2 diabetes.

Outpatient assessment of Karlsburg Diabetes Management System-based decision support.

OBJECTIVE: We sought to assess the benefit of the Karlsburg Diabetes Management System (KADIS) in conjunction with the continuous glucose monitoring system (CGMS) in an outpatient setting. RESEARCH DESIGN AND METHODS: A multicentric trial was performed in insulin-treated outpatients (n = 49), aged 21-70 years, with a mean diabetes duration of 14.2 years. Subjects were recruited from five outpatient centers and randomized for CGMS- or CGMS/KADIS-based decision support and followed up for 3 months. After two CGMS monitorings, the outcome parameters A1C (%), mean sensor glucose of the CGMS profile (MSG) (mmol/l), and duration of hyperglycemia (h/day) were evaluated. RESULTS: In contrast with the CGMS group (0.27 +/- 0.67%), mean change in A1C decreased in the CGMS/KADIS group during the follow-up (-0.34 +/- 0.49%; P < 0.01). MSG levels were not affected in the CGMS group (7.75 +/- 1.33 vs. 8.45 +/- 2.46 mmol/l) but declined in the CGMS/KADIS group (8.43 +/- 1.33 vs. 7.59 +/- 1.47 mmol/l; P < 0.05). Net KADIS effect (-0.60 [95% CI -0.96 to - 0.25%]; P < 0.01) was associated with reduced duration of hyperglycemia (4.6 vs. 1.0 h/day; P < 0.01) without increasing hypoglycemia. Multiple regression revealed that the A1C outcome was dependent on KADIS-based decision support. Age, sex, physician's specialty, diabetes type, and BMI had no measurable effect. CONCLUSIONS: If physicians were supported by CGMS/KADIS in therapeutic decisions, they achieved better glycemic control for their patients compared with support by CGMS alone. KADIS is a suitable decision support tool for physicians in outpatient diabetes care and has the potential to improve evidence-based management of diabetes.

Chronic hyperglycemia but not glucose variability determines HbA1c levels in well-controlled patients with type 2 diabetes.

To determine the relationships between HbA1c, characteristics of hyperglycemia and glycemic variability in well-controlled type 2 diabetes (HbA1c<7.0%), we studied 63 primary-care patients (36 men and 27 women), aged 34-75 years, with type 2 diabetes for 2-32 years using a continuous glucose monitoring system (CGMS) and standardized meal test (MMT). Duration of hyperglycemia (>8.0 mmol/l), standard deviation score (S.D.-score) and mean amplitude of glycemic excursions (MAGE) were analyzed from CGMS data and postprandial glucose during MMT (PPG(MMT)). Patients were hyperglycemic for 5.7h/day (median), experienced 4.1 hyperglycemic episodes/day, and 78% exceeded PPG levels of 8.0 mmol/l. HbA1c, though associated with the extent of hyperglycemia (r=0.40, p<0.001), failed to correlate with S.D.-score and MAGE. Multiple regression analysis demonstrated that HbA1c was predicted only by fasting glucose (R(2)=0.24, p<0.001) but neither by PPG(MMT), duration of hyperglycemia, S.D.-score nor MAGE. CGMS and meal test provide the tools for complete characterization of glycemia in type 2 diabetes. In well-controlled type 2 diabetes, HbA1c correlates with chronic hyperglycemia but not with glucose variability. Our data suggest that chronic sustained hyperglycemia and glucose fluctuations are two independent components of dysglycemia in diabetes.

Telemedicine-based KADIS combined with CGMS has high potential for improving outpatient diabetes care.

BACKGROUND: The Karlsburg Diabetes Management System (KADIS) was developed over almost two decades by modeling physiological glucose-insulin interactions. When combined with the telemedicine-based communication system TeleDIAB and a continuous glucose monitoring system (CGMS), KADIS has the potential to provide effective, evidence-based support to doctors in their daily efforts to optimize glycemic control. METHODS: To demonstrate the feasibility of improving diabetes control with the KADIS system, an experimental version of a telemedicine-based diabetes care network was established, and an international, multicenter, pilot study of 44 insulin-treated patients with type 1 and 2 diabetes was performed. Patients were recruited from five outpatient settings where they were treated by general practitioners or diabetologists. Each patient underwent CGMS monitoring under daily life conditions by a mobile monitoring team of the Karlsburg diabetes center at baseline and 3 months following participation in the KADIS advisory system and telemedicine-based diabetes care network. The current metabolic status of each patient was estimated in the form of an individualized "metabolic fingerprint." The fingerprint characterized glycemic status by KADIS-supported visualization of relationships between the monitored glucose profile and causal endogenous and exogenous factors and enabled evidence-based identification of "weak points" in glycemic control. Using KADIS-based simulations, physician recommendations were generated in the form of patient-centered decision support that enabled elimination of weak points. The analytical outcome was provided in a KADIS report that could be accessed at any time through TeleDIAB. The outcome of KADIS-based support was evaluated by comparing glycosylated hemoglobin (HbA1c) levels and 24-hour glucose profiles before and after the intervention. RESULTS: Application of KADIS-based decision support reduced HbA1c by 0.62% within 3 months. The reduction was strongly related to the level of baseline HbA1c, diabetes type, and outpatient treatment setting. The greatest benefit was obtained in the group with baseline HbA1c levels >9% (1.22% reduction), and the smallest benefit was obtained in the group with baseline HbA1c levels of 6-7% (0.13% reduction). KADIS was more beneficial for patients with type 1 diabetes (0.79% vs 0.48% reduction) and patients treated by general practitioners (1.02% vs 0.26% reduction). Changes in HbA1c levels were paralleled by changes in mean daily 24-hour glucose profiles and fluctuations in daily glucose. CONCLUSION: Application of KADIS in combination with CGMS and the telemedicine-based communication system TeleDIAB successfully improved outpatient diabetes care and management.

Effects of fatty acids on hepatic amino acid catabolism and fibrinogen synthesis in young healthy volunteers.

Increased synthesis rate of fibrinogen, an independent risk factor for cardiovascular disease, was recently reported in obese insulin-resistant female adolescents with chronic elevated nonesterified fatty acids (NEFA). It is unknown whether a short-term change of NEFA concentrations controls hepatic fibrinogen synthesis. Therefore, 10 healthy male volunteers (24.5 +/- 3.3 yr, body mass index 23.5 +/- 2.9 kg/m2) were investigated in random order under basal and elevated NEFA for 8 h. Leucine metabolism, the fractional synthesis rates (FSR) of plasma fibrinogen, and endogenous urea production rates were measured during primed, continuous infusion of [1-13C]leucine and [15N2]urea, respectively. Plasma alpha-[13C]ketoisocaproic acid and [15N2]urea enrichment values were measured with GC-MS. Plasma fibrinogen was isolated with the beta-alanine method, and fibrinogen-related [13C]leucine enrichment was analyzed by GC-CIRMS. Lipofundin infusion and subcutaneous heparin tripled NEFA and triglycerides in the tests. Plasma glucose, circulating insulin, human C-peptide, and plasma glucagon were not changed by the study procedure. Fibrinogen FSR were significantly lower in tests with NEFA elevation (18.44 +/- 4.67%) than in control tests (21.48 +/- 4.32%; P < 0.05). Plasma fibrinogen concentrations measured were not significantly different (NEFA test subjects: 1.85 +/- 0.33, controls: 1.97 +/- 0.54 g/l). Parameters of leucine metabolism, such as leucine rate of appearance, leucine oxidation, and nonoxidative leucine disposal, were not influenced by NEFA elevation, and endogenous urea production remained unchanged. NEFA contributes to short-term regulation of fibrinogen FSR in healthy volunteers under unchanged hormonal status, leucine metabolism, and overall amino acid catabolism. Its contribution might be of relevance at least after fat-rich meals, counteracting by reduction of FSR the blood viscosity increase implied by hyperlipidemia.