Adherence to drug therapy in patients with heart failure associated with reduced ejection fractions (HFrEF) during cardiac rehabilitation and after 3 and 6 months.

INTRODUCTION: Heart failure represents a major challenge for healthcare systems worldwide. Rehabilitation is recommended as an important pillar of therapy for these patients, especially for those with reduced left ventricular ejection fraction (HFrEF: heart failure with reduced ejection fraction). METHODS: The data collected in this multi-center project provide information on the rates of patients with HFrEF who were treated in five German rehabilitation facilities and whether the patients adhered to drug therapy at 3-/6-month follow-up. The project was supported by an unrestricted grant from Novartis-Pharma-GmbH. RESULTS: The mean age of the 234 patients included was 63.4 ± 10.6 years and 78% were male. The mean LVEF was 31 ± 8% at admission and 36 ± 10% at discharge. Only 20.6% of the patients were assigned to rehabilitation with the main indication HF. The most frequent main indication was acute coronary syndrome (46.6%). A high proportion of patients was already on the recommended drug therapy upon admission (94% beta blockers, 100% angiotensin-effective drugs, 70% mineralocorticoid receptor antagonists, etc.). This was optimized, in particular by a higher proportion of patients treated with sodium-glucose cotransporter-2 inhibitors (35% admission vs. 45% discharge) and sacubitril/valsartan (49% admission vs. 64% discharge), which was further optimized during the 6-month follow-up (e.g., 50% SGLT2 inhibitors, 67% sacubitril/valsartan). DISCUSSION: These data illustrate the effect of rehabilitation in terms of optimizing drug therapy, which stabilized over the course of 6 months. Furthermore, only a few patients with the main diagnosis HFrEF are referred for cardiac rehabilitation, although it is an essential part of guideline-based therapy.

Gluc-HET, a complementary chick embryo model for the characterization of antidiabetic compounds.

Insulin resistance and ß cell failure are the main causes of elevated blood glucose levels in Type 2 diabetes mellitus (T2DM), a complex and multifactorial metabolic disease. Several medications to treat or reduce the symptoms of T2DM are used, including the injection of insulin and the application of insulin sensitizing or glucose production reducing drugs. Furthermore, the use of phytochemicals has attracted increasing attention for the therapy and prevention of T2DM. In order to identify and characterize antidiabetic compounds, efficient test systems are required. Here we present a modified chick embryo model (hens egg test, HET), which has originally been developed to determine the potential irritancy of chemicals, as a versatile tool for the characterization of phytochemicals with antidiabetic properties. We termed this modified assay variation Gluc-HET. More precisely, we determined the influence of variations in the incubation time of the fertilized eggs and studied the effects of different buffer parameters, such as the temperature, composition and volume, used for drug application. In addition, we tested several putative antidiabetic plant extracts, which have been identified in an in-vitro primary screening procedure, for their effectiveness in reducing blood glucose levels in-ovo. Taken together, our Gluc-HET model has proven to be a reliable and manageable system for the characterization of antidiabetic compounds.

Biomolecular Characterization of Putative Antidiabetic Herbal Extracts.

Induction of GLUT4 translocation in the absence of insulin is considered a key concept to decrease elevated blood glucose levels in diabetics. Due to the lack of pharmaceuticals that specifically increase the uptake of glucose from the blood circuit, application of natural compounds might be an alternative strategy. However, the effects and mechanisms of action remain unknown for many of those substances. For this study we investigated extracts prepared from seven different plants, which have been reported to exhibit anti-diabetic effects, for their GLUT4 translocation inducing properties. Quantitation of GLUT4 translocation was determined by total internal reflection fluorescence (TIRF) microscopy in insulin sensitive CHO-K1 cells and adipocytes. Two extracts prepared from purslane (Portulaca oleracea) and tindora (Coccinia grandis) were found to induce GLUT4 translocation, accompanied by an increase of intracellular glucose concentrations. Our results indicate that the PI3K pathway is mainly responsible for the respective translocation process. Atomic force microscopy was used to prove complete plasma membrane insertion. Furthermore, this approach suggested a compound mediated distribution of GLUT4 molecules in the plasma membrane similar to insulin stimulated conditions. Utilizing a fluorescent actin marker, TIRF measurements indicated an impact of purslane and tindora on actin remodeling as observed in insulin treated cells. Finally, in-ovo experiments suggested a significant reduction of blood glucose levels under tindora and purslane treated conditions in a living organism. In conclusion, this study confirms the anti-diabetic properties of tindora and purslane, which stimulate GLUT4 translocation in an insulin-like manner.

Degradable glycine-based photo-polymerizable polyphosphazenes for use as scaffolds for tissue regeneration.

Photo-polymerizable scaffolds are designed and prepared via short chain poly(organo)phosphazene building blocks bearing glycine allylester moieties. The polyphosphazene was combined with a trifunctional thiol and divinylester in various ratios, followed by thiol-ene photo-polymerization to obtain porous matrices. Degradation studies under aqueous conditions showed increasing rates in correlation with the polyphosphazene content. Preliminary cell studies show the non-cytotoxic nature of the polymers and their degradation products, as well as the cell adhesion and proliferation of adipose-derived stem cells.

Risk factors for, and prevalence of, sleep apnoea in cardiac rehabilitation facilities in Germany: The Reha-Sleep registry.

AIM: To determine the prevalence of, and the risk factors for, sleep apnoea in cardiac rehabilitation (CR) facilities in Germany. METHODS: 1152 patients presenting for CR were screened for sleep-disordered breathing with 2-channel polygraphy (ApneaLink™; ResMed). Parameters recorded included the apnoea-hypopnoea index (AHI), number of desaturations per hour of recording (ODI), mean and minimum nocturnal oxygen saturation and number of snoring episodes. Patients rated subjective sleep quality on a scale from 1 (poor) to 10 (best) and completed the Epworth Sleepiness Scale (ESS). RESULTS: Clinically significant sleep apnoea (AHI ≥15/h) was documented in 33% of patients. Mean AHI was 14 ± 16/h (range 0-106/h). Sleep apnoea was defined as being of moderate severity in 18% of patients (AHI ≥15-29/h) and severe in 15% (AHI ≥30/h). There were small, but statistically significant, differences in ESS score and subjective sleep quality between patients with and without sleep apnoea. Logistic regression model analysis identified the following as risk factors for sleep apnoea in CR patients: age (per 10 years) (odds ratio (OR) 1.51; p<0.001), body mass index (per 5 units) (OR 1.31; p=0.001), male gender (OR 2.19; p<0.001), type 2 diabetes mellitus (OR 1.45; p=0.040), haemoglobin level (OR 0.91; p=0.012) and witnessed apnoeas (OR 1.99; p<0.001). CONCLUSIONS: The findings of this study indicate that more than one-third of patients undergoing cardiac rehabilitation in Germany have sleep apnoea, with one-third having moderate-to-severe SDB that requires further evaluation or intervention. Inclusion of sleep apnoea screening as part of cardiac rehabilitation appears to be appropriate.

Identification of novel insulin mimetic drugs by quantitative total internal reflection fluorescence (TIRF) microscopy.

BACKGROUND AND PURPOSE: Insulin stimulates the transport of glucose in target tissues by triggering the translocation of glucose transporter 4 (GLUT4) to the plasma membrane. Resistance to insulin, the major abnormality in type 2 diabetes, results in a decreased GLUT4 translocation efficiency. Thus, special attention is being paid to search for compounds that are able to enhance this translocation process in the absence of insulin. EXPERIMENTAL APPROACH: Total internal reflection fluorescence (TIRF) microscopy was applied to quantify GLUT4 translocation in highly insulin-sensitive CHO-K1 cells expressing a GLUT4-myc-GFP fusion protein. KEY RESULTS: Using our approach, we demonstrated GLUT4 translocation modulatory properties of selected substances and identified novel potential insulin mimetics. An increase in the TIRF signal was found to correlate with an elevated glucose uptake. Variations in the expression level of the human insulin receptor (hInsR) showed that the insulin mimetics identified stimulate GLUT4 translocation by a mechanism that is independent of the presence of the hInsR. CONCLUSIONS AND IMPLICATIONS: Taken together, the results indicate that TIRF microscopy is an excellent tool for the quantification of GLUT4 translocation and for identifying insulin mimetic drugs.

Resting electrocardiogram predicts mortality in postmenopausal women with coronary heart disease or with risk factors for coronary heart disease.

BACKGROUND: Information about prognostic utility is limited for numerous electrocardiogram (ECG) abnormalities and is particularly scarce in women with coronary heart disease (CHD) or at increased risk of CHD occurrence. DESIGN: This study used a prospectively planned observational post-hoc analysis of a negative randomised trial designed for other purposes. METHODS: ECGs of 9789 postmenopausal women were analysed at a core laboratory. ECG abnormalities were determined and evaluated for mortality risk on top of established clinical risk factors and simultaneously with all other ECG abnormalities. RESULTS: During a median follow-up of 5.6 years, 500 women sustained coronary death. Normal versus abnormal baseline ECGs were associated with an annual rate of 0.53% versus 1.28% coronary deaths. Ten clinically common ECG abnormalities, including left atrial abnormality, fragmented QRS, and Cornell voltage-only left ventricular hypertrophy, emerged as independent significant predictors of coronary death, eight of them also predicted all-cause mortality. Each ECG abnormality acted as a risk multiplier for a coexisting ECG abnormality. Two or three of any of the ECG abnormalities simultaneously present on the baseline ECG identified subsets with hazard ratios for coronary death of 3.3 or 5.5 respectively. Consideration of ECG abnormalities significantly improved risk stratification by common clinical parameters. CONCLUSION: The presence of ECG abnormalities provides independent risk information over and above that of established risk factors, both for women with CHD or at increased risk of occurrence of CHD. Normal ECG assures a low mortality risk regardless of whether CHD exists or not. The data derived could be applied to corresponding postmenopausal women in daily clinical practice.

PBTK modelling platforms and parameter estimation tools to enable animal-free risk assessment: recommendations from a joint EPAA--EURL ECVAM ADME workshop.

Information on toxicokinetics is critical for animal-free human risk assessment. Human external exposure must be translated into human tissue doses and compared with in vitro actual cell exposure associated to effects (in vitro-in vivo comparison). Data on absorption, distribution, metabolism and excretion in humans (ADME) could be generated using in vitro and QSAR tools. Physiologically-based toxicokinetic (PBTK) computer modelling could serve to integrate disparate in vitro and in silico findings. However, there are only few freely-available PBTK platforms currently available. And although some ADME parameters can be reasonably estimated in vitro or in silico, important gaps exist. Examples include unknown or limited applicability domains and lack of (high-throughput) tools to measure penetration of barriers, partitioning between blood and tissues and metabolic clearance. This paper is based on a joint EPAA--EURL ECVAM expert meeting. It provides a state-of-the-art overview of the availability of PBTK platforms as well as the in vitro and in silico methods to parameterise basic (Tier 1) PBTK models. Five high-priority issues are presented that provide the prerequisites for wider use of non-animal based PBTK modelling for animal-free chemical risk assessment.

Acetaminophen changes intestinal epithelial cell membrane properties, subsequently affecting absorption processes.

BACKGROUND/AIMS: Acetaminophen (APAP) effects on intestinal barrier properties are less investigated. APAP may lead to a changed bioavailability of a subsequently administered drug or diet in the body. We investigated the influence of APAP on enterocytic cell membrane properties that are able to modify the net intestinal absorption of administered substances across the Caco-2 barrier model. METHODS: The effect of APAP on cytotoxicity was measured by LDH assay, TER value and cell capacitance label-free using impedance monitoring, membrane permeability by FITC-dextrans, and efflux transporter MDR1 activity by Rh123. APAP levels were determined by HPLC analysis. Cell membrane topography and microvilli were investigated using SEM and intestinal alkaline phosphatase (Alpi) and tight junction protein 1 (TJP1) expression by western blot analysis. RESULTS: APAP changed the apical cell surface, reduced the number of microvilli and protein expression of Alpi as a brush border marker and TJP1, increased the membrane integrity and concurrently decreased cell capacitance over time. In addition, APAP decreased the permeability to small molecules and increased the efflux transporter activity, MDR1. CONCLUSION: APAP alters the Caco-2 cell membrane properties by different mechanisms and reduces the permeability to administered substances. These findings may help to optimize therapeutic implications.

Reconstructed human epidermis for skin absorption testing: results of the German prevalidation study.

Exposure to chemicals absorbed by the skin can threaten human health. In order to standardise the predictive testing of percutaneous absorption for regulatory purposes, the OECD adopted guideline 428, which describes methods for assessing absorption by using human and animal skin. In this study, a protocol based on the OECD principles was developed and prevalidated by using reconstructed human epidermis (RHE). The permeation of the OECD standard compounds, caffeine and testosterone, through commercially available RHE models was compared to that of human epidermis and animal skin. In comparison to human epidermis, the permeation of the chemicals was overestimated when using RHE. The following ranking of the permeation coefficients for testosterone was obtained: SkinEthic > EpiDerm, EPISKIN > human epidermis, bovine udder skin, pig skin. The ranking for caffeine was: SkinEthic, EPISKIN > bovine udder skin, EpiDerm, pig skin, human epidermis. The inter-laboratory and intra-laboratory reproducibility was good. Long and variable lag times, which are a matter of concern when using human and pig skin, did not occur with RHE. Due to the successful transfer of the protocol, it is now in the validation process.

ST resolution in a single electrocardiographic lead: a simple and accurate predictor of cardiac mortality in patients with fibrinolytic therapy for acute ST-elevation myocardial infarction.

BACKGROUND: The extent of ST-segment resolution in the 12-lead electrocardiograph (ECG) obtained early after reperfusion therapy in patients with ST-elevation myocardial infarction (MI) has been shown to predict short- and long-term mortalities. To improve the ease of this method in clinical practice, we sought to evaluate the optimal cutoffs and the prognostic value of ST resolution (STR) measured in a single ECG lead. METHODS: In conjunction with the Intravenous nPA for the Treatment of Infarcting Myocardium Early (InTIME)-2 study, in which patients with an ST-elevation MI of <6 hours' duration were treated with alteplase or lanoteplase, 12-lead ECGs were obtained at baseline and 90 minutes after the start of fibrinolytic therapy in 3030 patients. RESULTS: There was a close correlation between the extent of the sum STR and single-lead ST-elevation resolution ( r = 0.94). The optimal cutoffs for definition of single-lead complete, partial, and no-STR groups were 70% and 50% for anterior infarcts and 70% and 20% for inferior infarcts. The cardiac 30-day mortality rates for the 2 sets of risk groups by sum or single-lead STR were as follows: no resolution, 9.5% vs 10.3%; partial resolution, 5.0% vs 3.6%; complete resolution, 2.0% vs 1.2%. The predictive power was significantly better for single-lead STR. CONCLUSIONS: ST resolution obtained in a single lead is an easy and accurate prognosticator of cardiac 30-day mortality in patients with ST-elevation MI. It is therefore useful for early identification of low- and high-risk subgroups after fibrinolysis and as a surrogate end point in clinical trials.

Kiwi fruit (Actinidia chinensis L.) polysaccharides exert stimulating effects on cell proliferation via enhanced growth factor receptors, energy production, and collagen synthesis of human keratinocytes, fibroblasts, and skin equivalents.

Within physiological engineering exogenous carbohydrates were recently confirmed as pharmacologically active compounds. To investigate potential dermatological activity purified polysaccharides from kiwi fruits (Actinidia chinensis L., Actinidiaceae) were characterized concerning monomer composition, linkage types and molecular weights and were tested under in vitro conditions for regulating activities on cell physiology of human keratinocytes, fibroblasts, and skin equivalents. Ten micrograms per milliliter of raw polysaccharide, neutral type-II-arabinogalactans, and acidic arabinorhamnogalacturonans of kiwi fruits stimulated cell proliferation of human keratinocytes (NHK, HaCaT) up to 30% significantly while mitochondrial activity was stimulated for nearly 25% in regard to control cells. Fibroblasts were not as sensitive as keratinocytes but >130 microg/ml kiwi fruit polysaccharides increased proliferation and ATP-synthesis significantly, too. Proliferation-stimulating activity was dependent on terminal 1-alpha-l-arabinose residues since enzymatic release of these sugar moieties caused significantly decreased proliferation of HaCaT and fibroblasts of about 10% in regard to untreated cells. In three dimensional skin equivalents, it was shown that the polysaccharides led to a doubled collagen synthesis of fibroblasts compared to the normally strongly reduced biosynthetic activity.

In vitro skin irritation: facts and future. State of the art review of mechanisms and models.

The skin is the main target tissue for exogenous noxes, protecting us from harmful environmental hazards, UV-irradiation and endogenous water loss. It is composed of three layers, whereas the outermost epidermis is a squamous epithelium that mainly consists of keratinocytes. These cells execute a terminal differentiation, which finally results in the assembly of the stratum corneum. This layer, consisting of cornified keratinocytes, is an effective barrier against a vast number of substances. Apart of this, keratinocytes play crucial roles in the immune surveillance and the initiation, modulation and regulation of inflammation in the epidermis. Regarding cutaneous inflammatory reactions, skin irritation is one of the most common adverse effect in humans. For reasons of human safety assessment new chemicals are still evaluated for irritant potentials by application to animals followed by visible changes such as erythema and oedema. Testing for skin irritation in animals potentially cause them pain and discomfort. Furthermore, the results are not always predictive for those found in humans. In order to replace animal testing and to improve the prediction of irritants, the cosmetic and toiletry industry, in Europe represented by Colipa, develops and uses several alternative in vitro test systems. In this respect, the use of in vitro reconstructed organotypic skin equivalents are mostly favored, because of their increasingly close resemblance to human skin. Due to ethical and scientific questions and on account of the 7th amendment of the European Council Directive 76/768/EEC, the authors see the requirement to drive the development of alternative tests for irritants. Therefore, this article centres on cosmetic ingredients and provides the readership an overview of the state of art of cellular mechanisms of skin irritation and summarizes the results of the commonly used skin equivalents to evaluate irritation in vitro.