A novel and selective sodium-glucose cotransporter-2 inhibitor, tofogliflozin, improves glycaemic control and lowers body weight in patients with type 2 diabetes mellitus.

AIM: To assess the efficacy, safety and tolerability of different doses of tofogliflozin, a novel, highly selective sodium-glucose cotransporter 2 (SGLT2) inhibitor, in patients with type 2 diabetes mellitus (T2DM). METHODS: In a 12-week, multicentre, multinational, randomized, double-blind, parallel-group, placebo-controlled, dose-finding study, patients with inadequate glycaemic control from diet and exercise alone, or from diet and exercise plus a stable dose of metformin, were randomized to one of five doses of tofogliflozin (2.5, 5, 10, 20, or 40 mg) or placebo. The primary efficacy endpoint was absolute change at week 12 from baseline in glycated haemoglobin (HbA1c), minus the change in the placebo group. RESULTS: Statistically significant dose-dependent reductions in HbA1c were shown in all treated groups except the 2.5-mg dose group, with a maximum reduction of 0.56% (placebo-subtracted) at the 40-mg dose, along with increased urinary glucose excretion. Metformin treatment had no substantial influence on tofogliflozin efficacy. Dose-dependent reductions in fasting plasma glucose and body weight were observed, and glucose intolerance was improved, with a trend towards blood pressure reduction. Slight increases were observed for mean ketone bodies with no abnormal change in ketone body ratio. No deaths or treatment-related serious adverse events were reported. The incidence of adverse events was similar in the placebo (37.9%) to that in the tofogliflozin group (35.9-46.3%). Withdrawal because of adverse events was rare (≤2 patients per treatment group), with similar rates of withdrawal in the placebo and tofogliflozin groups. CONCLUSIONS: A once-daily dose of tofogliflozin for 12 weeks was an effective, safe and well-tolerated treatment for T2DM.

C.E.R.A. safety profile: a pooled analysis in patients with chronic kidney disease.

BACKGROUND: C.E.R.A., a continuous erythropoietin receptor activator, is a long-acting erythropoiesis-stimulating agent (ESA) that is approved for the treatment of renal anemia. This analysis evaluated the safety profile of C.E.R.A. in comparison to that of other ESAs in patients with chronic kidney disease (CKD). METHODS: Safety parameters were analyzed in a pooled population comprising all patients with CKD on dialysis and not on dialysis from the completed Phase II and Phase III studies in the C.E.R.A. clinical program (Phase II/III population); patients were treated with either C.E.R.A. (n = 1,789) or comparator ESA (n = 948). Differences between treatment groups in safety parameters were identified by either a 2% difference in incidence between groups, or a statistically significant difference between groups (p < or = 0.05 with the Fisher's exact test, which was used as a conservative screening tool). To assess changes in safety findings over time, long-term safety data were analyzed from patients who were given the option to enter long-term safety studies upon completing their initial Phase II/III study (safety extension population). RESULTS: Compared with the C.E.R.A. group, the incidence of adverse events (AEs) was higher in the comparator ESA group in the Phase II/III population (C.E.R.A. vs. comparator ESA, 89.5% vs. 91.8%, p = 0.067), and significantly so in the safety extension population (93.0% vs. 95.8%, p = 0.003). The incidence of serious AEs was significantly higher in the comparator ESA group than in the C.E.R.A. group in both analysis populations (Phase II/III population, 37.8% vs. 42.4%, p = 0.021; safety extension population, 53.3% vs. 59.7%, p = 0.001). However, there was no consistent pattern of clinical events that could explain these differences between the treatment groups. CONCLUSION: Analysis of safety events in patients with renal anemia receiving long-term treatment with C.E.R.A. shows a safety profile comparable to that of other ESAs.

Comprehensive identification of genes driven by ERV9-LTRs reveals TNFRSF10B as a re-activatable mediator of testicular cancer cell death.

The long terminal repeat (LTR) of human endogenous retrovirus type 9 (ERV9) acts as a germline-specific promoter that induces the expression of a proapoptotic isoform of the tumor suppressor homologue p63, GTAp63, in male germline cells. Testicular cancer cells silence this promoter, but inhibitors of histone deacetylases (HDACs) restore GTAp63 expression and give rise to apoptosis. We show here that numerous additional transcripts throughout the genome are driven by related ERV9-LTRs. 3' Rapid amplification of cDNA ends (3'RACE) was combined with next-generation sequencing to establish a large set of such mRNAs. HDAC inhibitors induce these ERV9-LTR-driven genes but not the LTRs from other ERVs. In particular, a transcript encoding the death receptor DR5 originates from an ERV9-LTR inserted upstream of the protein coding regions of the TNFRSF10B gene, and it shows an expression pattern similar to GTAp63. When treating testicular cancer cells with HDAC inhibitors as well as the death ligand TNF-related apoptosis-inducing ligand (TRAIL), rapid cell death was observed, which depended on TNFRSF10B expression. HDAC inhibitors also cooperate with cisplatin (cDDP) to promote apoptosis in testicular cancer cells. ERV9-LTRs not only drive a large set of human transcripts, but a subset of them acts in a proapoptotic manner. We propose that this avoids the survival of damaged germ cells. HDAC inhibition represents a strategy of restoring the expression of a class of ERV9-LTR-mediated genes in testicular cancer cells, thereby re-enabling tumor suppression.

Synthesis and in vitro efficacy of transferrin conjugates of the anticancer drug chlorambucil.

One strategy for improving the selectivity and toxicity profile of antitumor agents is to design drug carrier systems employing soluble macromolecules or carrier proteins. Thus, five maleimide derivatives of chlorambucil were bound to thiolated human serum transferrin which differ in the stability of the chemical link between drug and spacer. The maleimide ester derivatives 1 and 2 were prepared by reacting 2-hydroxyethylmaleimide or 3-maleimidophenol with the carboxyl group of chlorambucil, and the carboxylic hydrazone derivatives 5-7 were obtained through reaction of 2-maleimidoacetaldehyde, 3-maleimidoacetophenone, or 3-maleimidobenzaldehyde with the carboxylic acid hydrazide derivative of chlorambucil. The alkylating activity of transferrin-bound chlorambucil was determined with the aid of 4-(4-nitrobenzyl)pyridine (NBP) demonstrating that on average 3 equivalents were protein-bound. Evaluation of the cytotoxicity of free chlorambucil and the respective transferrin conjugates in the MCF7 mammary carcinoma and MOLT4 leukemia cell line employing a propidium iodide fluorescence assay demonstrated that the conjugates in which chlorambucil was bound to transferrin through non-acid-sensitive linkers, i.e., an ester or benzaldehyde carboxylic hydrazone bond, were not, on the whole, as active as chlorambucil. In contrast, the two conjugates in which chlorambucil was bound to transferrin through acid-sensitive carboxylic hydrazone bonds were as active as or more active than chlorambucil in both cell lines. Especially, the conjugate in which chlorambucil was bound to transferrin through an acetaldehyde carboxylic hydrazone bond exhibited IC50 values which were approximately 3-18-fold lower than those of chlorambucil. Preliminary toxicity studies in mice showed that this conjugate can be administered at higher doses in comparison to unbound chlorambucil. The structure-activity relationships of the transferrin conjugates are discussed with respect to their pH-dependent acid sensitivity, their serum stability, and their cytotoxicity.

Drug-polymer conjugates containing acid-cleavable bonds.

Drug-polymer conjugates are potential candidates for the selective delivery of anticancer agents to tumor tissue. Incorporating acid-sensitive bonds between the drug and the polymer is an attractive approach because it ensures effective release of the polymer-bound drug at the tumor site. This release is either extracellular, resulting from the slightly acidic pH in tumor tissue, or intracellular, in acidic endosomes or lysosomes after cellular uptake of the drug-polymer conjugate. This paper reviews acid-sensitive drug-polymer conjugates developed during the past 20 years and outlines aspects for further development in this research field.

Calibration of the viscometric glucose sensor before its use in physiological liquids--compensation for the colloid-osmotic effect.

The function of the recently described viscometric affinity sensor (VAS), which measures glucose due to its strong effect on the viscosity of a sensitive liquid containing Concanavalin A (ConA) and dextran, was analysed for osmotic and colloid-osmotic effects on the glucose reading. The suction of low- and high-molecular weight osmotica on the membrane of the microdialysis fibre was measured using a membrane osmometer built from the microdialysis probe of the VAS. The reduction of the sensor read-out in blood plasma can be completely explained by a change in small osmotic volume fluxes through the dialysis membrane, which affect the ConA concentration and the viscosity after the flow of the sensitive liquid through the dialysis probe. The measuring error could be prevented by the presence of the polyethylene glycol 6000 at an isotonic concentration in the glucose standard solutions used for sensor calibration.

Compensation of temperature and concanavalin A concentratration effects for glucose determination by the viscometric affinity assay.

A viscometer suitable for rapid measurements in small volumes of highly viscous liquids is described. Using this device the viscometric affinity assay for glucose was studied under variable conditions in order to obtain basic information for the design of a viscometric glucose sensor. The viscosity of the dextran/Concanavalin A (ConA) solution is sensitive to glucose in a broad range of the shear stress. However, for measuring the glucose concentration with this sensitive liquid the strong dependence of the absolute viscosity on temperature and ConA concentration has to be taken into account. For the purpose of calibration a parameter more suitable than the absolute viscosity is the relative fluidity (F(r)) that is defined by the actual measured viscosity at a given glucose concentration, the reference viscosity at a standard glucose concentration, and a constant linearization coefficient. F(r) shows a linear dependence on the glucose concentration in the therapeutically interesting range up to 30 mM and is not significantly changed by moderate variations of the ConA concentration or temperature.

Transferrin conjugates of doxorubicin: synthesis, characterization, cellular uptake, and in vitro efficacy.

One strategy for improving the antitumor selectivity and toxicity profile of antitumor agents is to design drug carrier systems employing suitable carrier proteins. Thus, thiolated human serum transferrin was conjugated with four maleimide derivatives of doxorubicin that differed in the stability of the chemical link between drug and spacer. Of the maleimide derivatives, 3-maleimidobenzoic or 4-maleimidophenylacetic acid was bound to the 3'-amino position of doxorubicin through a benzoyl or phenylacetyl amide bond, and 3-maleimidobenzoic acid hydrazide or 4-maleimidophenylacetic acid hydrazide was bound to the 13-keto position through a benzoyl hydrazone or phenylacetyl hydrazone bond. The acid-sensitive transferrin conjugates prepared with the carboxylic hydrazone doxorubicin derivatives exhibited an inhibitory efficacy in the MDA-MB-468 breast cancer cell line and U937 leukemia cell line comparable to that of the free drug (employing the BrdU (5-bromo-2'-deoxyuridine) incorporation assay and tritiated thymidine incorporation assay, respectively, IC50 approximately 0.1-1 mM), whereas conjugates with the amide derivatives showed no activity. Furthermore, antiproliferative activity of the most active transferrin conjugate (i.e. the conjugate containing a benzoyl hydrazone link) was demonstrated in the LXFL 529 lung carcinoma cell line employing a sulforhodamine B assay. In contrast to in vitro studies in tumor cells, cell culture experiments performed with human endothelial cells (HUVEC) showed that the acid-sensitive transferrin conjugates of doxorubicin were significantly less active than free doxorubicin (IC50 values approximately 10-40 higher by the BrdU incorporation assay), indicating selectivity of the doxorubicin-transferrin conjugates for tumor cells. Fluorescence microscopy studies in the MDA-MB-468 breast cancer cell showed that free doxorubicin accumulates in the cell nucleus, whereas doxorubicin of the transferrin conjugates is found localized primarily in the cytoplasm. The differences in the intracellular distribution between transferrin-doxorubicin conjugates and doxorubicin were confirmed by laser scanning confocal microscopy in LXFL 529 cells after a 24 h incubation that revealed an uptake and mode of action other than intercalation with DNA. The relationship between stability, cellular uptake, and cytotoxicity of the conjugates is discussed.

Indirect myocardial revascularization--an experimental study in the dog.

It has been shown in previous studies that grafting a free skeletal muscle flap onto the heart of the dog produces myocardial revascularization. In order to evaluate the functional aspect of these neovessels, an Ameroid constrictor was implanted into the LADs (left anterior descending coronary artery) of five dogs. At the same time, the free muscle flap was grafted onto the anterior wall of the heart. The arterial anastomosis was achieved with the internal mammary artery. Venous flow was drained into the right atrium. About 12 months later, the regional myocardial blood flow was evaluated adopting the radioactive microsphere technique. This was carried out with the LAD occluded by the Ameroid constrictor and the circumflex artery (CX) open (control conditions). Then the CX was occluded in order to exclude collateral blood flow. Afterwards another occlusion of the CX was performed ("preconditioning") to produce maximal opening of the anatomical collaterals. It was found that regional myocardial blood flow was unchanged in the antero-lateral parts of the hearts despite complete occlusion of the coronaries supplying this area. In the parts supplied by the CX, though, flow was minimal. "Preconditioning" positively influenced the perfusion of this region only in the epicardial parts. Histologically large sinusoid-like blood conductors connected the graft and the heart. Thus, grafting a free skeletal muscle flap onto the heart in the dog produces functional myocardial revascularization in the dog.

A cardiac assist device as blood supply in ischemic heart disease.

Before the event of modern bypass surgery revascularisation of the heart was tried with the transplantation of pedicled muscle flaps onto the heart. For patients who cannot undergo bypass surgery this method could be--in a modified way--of help. This was shown in 12 dogs. In those a free muscle flap was transplanted onto the heart which had undergone a previous artificial infarction. The muscle flap was anastomosed with the a. mammaria, venous flow was directed into the right atrium. After 5 months the muscle was almost completely atrophic, but a fit capillary network was penetrating the underlying myocardium.

Serum proteins as drug carriers of anticancer agents: a review.

Targeted delivery of anticancer drugs is one of the most actively pursued goals in anticancer chemotherapy. Serum proteins such as transferrin, albumin, and low-density lipoprotein (LDL) offer promise for the selective delivery of antineoplastic agents due to their accumulation in tumor tissue. Uptake of these proteins in solid tumors is mediated by a number of factors, including an increased metabolic activity of tumors, an enhanced vascular permeability of tumor blood vessels for circulating macromolecules, and a lack of a functional lymphatic drainage system in tumor tissue. At the tumor site, transferrin, low-density lipoprotein, and albumin are taken up by the tumor cell through receptor-mediated and fluid phase endocytosis, respectively. Serum protein conjugates can be designed to release the bound antitumor drug after cellular uptake of the drug conjugate. This review covers the diagnostic evidence for tumor accumulation of serum proteins and the design, development, and biological evaluation of drug conjugates with transferrin, albumin, and low-density lipoprotein.

[Neo-angiogenesis after transplantation of a free skeletal muscle flap to the myocardium of the dog]. / Neoangiogenese nach Transplantation eines freien Skelettmuskellappens auf das Myokard des Hundes.

For the study of the process of neovascularization the effects of the transplantation of a free skeletal muscle flap on the heart of the dog were studied. For this purpose, in 14 mongrel dogs a myocardial infarction of the anterior wall of the heart was produced by the selective injection of microspheres into the left anterior descending artery. In 12 of those, on the area of infarction 4 weeks later a free pectoralis muscle flap was transplanted with its pars anterior in contact with the myocardium (group A). The arterial anastomosis was achieved with the internal mammary artery, the venous flow directed into the right atrium. In 2 other dogs (group B) the pectoralis flap was transplanted with irt pars anterior on a healthy myocardium. In 2 further dogs (group C) the pectoralis flap was transplanted on a infarcted myocardium with its pars posterior, i.e. the muscle fascia interponed. 16 weeks later a microcorrosion-cast-preparation and histological examinations showed in group A and B a prominent capillary network penetrating from the muscle into the myocardium, whereas in group C the muscle fascia inhibited this process. Thus, for the process of neovascularization the "bloody" contact between a skeletal muscle graft and the myocardium is necessary. Results are discussed with regard to possible factors inducing neoangiogenesis.

Cardiac arrhythmias during experimental induction and management of myocardial infarction in the dog.

The experimental production of a myocardial infarction (MI) in the dog was achieved adopting a new catheter technique. After induction of the MI, a variety of arrhythmias appeared and were classified according to the Lown-classification as more or less severe. The therapy was achieved with antiarrhythmics class 1-111 (Vaughn-Williams classification) for late premature ventricular beats or couplets. Sinus tachycardia was often terminated by occular or sinus carotis pressure or a new selective sinus blocking agent. Results showed Amiodarone to be the drug of choice in the treatment of severe arrhythmias (Lown IV and V) like triplets or salves of extrasystolies. Ventricular fibrillation always resulted in the death of the animal, because fibrillation was not convertible by direct current cardioversion, endovenous injection of Lidocaine or even internal cardiac massage. The registration of hemodynamic parameters (left ventricular end diastolic pressure, wedge pressure, pulmonary and aortic pressure) was shown to be important in controlling the therapy, as well as blood gas and electrolyte analysis.

The alpha/beta carboxy-terminal domains of p63 are required for skin and limb development. New insights from the Brdm2 mouse which is not a complete p63 knockout but expresses p63 gamma-like proteins.

p63, an ancestral transcription factor of the p53 family, has three C-terminal isoforms whose relative in vivo functions are elusive. The p63 gene is essential for skin and limb development, as vividly shown by two independent global knockout mouse models. Both strains, although constructed differently, have identical and severe phenotypes, characterized by absent epidermis and hindlimbs and only rudimentary forelimbs at birth. Here we show that mice from one model, Brdm2, express normal levels of truncated p63 proteins that contain the DNA binding and oligomerization domain but lack the long carboxy-terminal SAM (sterile alpha-motif) and post-SAM domains that are specific for the alpha and beta isoforms. As such, transcriptionally active p63 proteins from Brdm2 mice resemble the naturally occurring p63gamma isoforms, which of all the p63 isoforms most closely resemble p53. Thus, Brdm2 mice are p63alpha/beta isoform-specific knockout mice, gaining unexpected new importance. Our studies identify that p63alpha/beta but not p63gamma are absolutely required for proper skin and limb development.

[Perinatal problems in fetal macrosomy and its relation to maternal Hb A1C]. / Perinatale Problematik bei kindlicher Makrosomie und ihre Beziehung zum mütterlichen HbA1C.

Although the frequency of macrosomic newborns decreased within the last years due to an intensive centralized care of women with diabetes mellitus, 175 new borns had a birth weight of more than 4,000 grams in 1986 (total number of deliveries 2,339). Within this collective increasing maternal as well as fetal risks and perinatal complications increased rapidly corresponding to the rising birth weight. When clinical parameters were related to maternal levels of HBA1C, which was determined immediately after delivery, a dependency of the height of glycolised hemoglobin was related to several perinatal problems e.g. maternal obesity, gestosis, maternal-fetal disproportion, operative delivery and fetal morbidity. These data stress upon the necessity of a general screening program for the early detection of metabolic dysfunction of carbohydrate.

Systemic Fusarium oxysporum infection in an immunocompetent patient with an adult respiratory distress syndrome (ARDS) and extracorporal membrane oxygenation (ECMO).

We report a case of disseminated fusariosis in a 42-year-old patient with adult respiratory distress syndrome (ARDS) and extracorporal membrane oxygenation (ECMO), but without definite immunosuppression. Fusarium oxysporum was isolated from a bronchial lavage taken 6 days ante mortem. Despite antifungal treatment with amphotericin B and flucytosine the patient died in septic multiorgan failure. A post-mortem examination was performed. The patient's liver was found to contain fungus cells and F. oxysporum could be cultured from ascites.

[Inhaled nitric monoxide. Application and continuous measurement of concentration]. / Inhaliertes Stickstoffmonoxid. Anwendung und kontinuierliche Konzentrationsmessung.

Inhaled nitric oxide (NO) is a selective pulmonary vasodilator that may be useful in the treatment of patients with severe pulmonary hypertension. We describe a delivery system of inhaled NO that allows safe application and continuous measurement of the inspired NO concentration during mechanical ventilation. From a gas cylinder containing NO in N2 (600 ppm NO), an adjustable amount of gas is introduced into the inspiratory side of the tubing system via a pressure reduction valve, a magnetic valve, and a special injection nozzle. The NO concentration is diluted to the desired value by the tidal volume. The magnetic valve is connected to the ventilator and opens at the beginning of each inspiration and closes after a predetermined time. The gas volume is proportional to the pressure at the magnetic valve and the opening time. To monitor the inspiratory NO concentration, a specimen of gas is taken from an angle-connector and passed over an electrochemical sensor. The second nozzle of the sensor is connected to a water seal, which is adjusted to the positive end-expiratory pressure level of the ventilator to insure that the gas flow over the sensor is limited to inspiration.

Large increase in cardiac output in a patient with ARDS and acute right heart failure during inhalation of nitric oxide.

BACKGROUND: Inhaled nitric oxide (NO), a selective pulmonary vasodilator, reduces pulmonary artery pressure in patients with acute respiratory distress syndrome (ARDS). In spite of the reduction of right ventricular afterload, the effect of NO on cardiac output remains unclear. METHODS: A patient with ARDS and echocardiographically determined severe acute right heart failure was treated with increasing concentrations of inhaled nitric oxide (NO). Haemodynamic and gas exchange variables were determined for each concentration of NO. NO treatment was continued for 3 days. RESULTS: During initial right heart failure, administration of NO resulted in a large increase (32%) in cardiac output in a dose-dependent manner. When right ventricular function had improved, inhalation of NO did not increase cardiac output. CONCLUSION: Our observations suggest that inhalation of NO is likely to increase cardiac output in ARDS when severe acute right heart failure is present.