LAPS Insulin115: A novel ultra-long-acting basal insulin with a unique action profile.

AIMS: To conduct a comprehensive pre-clinical study of the novel ultra-long acting insulin analogue LAPS Insulin115. METHODS: Pharmacokinetic/pharmacodynamic studies comparing LAPS Insulin115 with other basal insulins were conducted in genetically diabetic (db/db) mice. Insulin signalling in the major target organs was analysed using Western blot after single subcutaneous injection in wild-type male Wistar rats. Using in vitro assays we analysed transendothelial transport, insulin receptor (IR) interaction, and the mitogenic and metabolic properties of LAPS Insulin115. Furthermore, IR downregulation after long-term exposure to high concentrations of LAPS Insulin115 was analysed using an in vitro desensitization/resensitization model. RESULTS: The novel Fc-conjugated insulin derivative LAPS Insulin115 showed an extensively prolonged pharmacokinetic and pharmacodynamic profile in rodents. Despite its size of 59 kDa, LAPS Insulin115 passes the vascular endothelial barrier and induces insulin signalling in all major target tissues in rats. In vitro, LAPS Insulin115 showed a very slow onset of action because of its reduced IR affinity; however, after long-term stimulation it was equipotent in respect to its metabolic potency and showed no increased mitogenic action when compared with regular insulin. Remarkably, under conditions of chronic exposure, LAPS Insulin115 does not induce irreversible desensitization of target cells, which is probably attributable to much less prominent IR downregulation. CONCLUSION: Thus, LAPS Insulin115 exhibits a unique in vivo and in vitro profile and thereby represents an excellent candidate for a once-weekly insulin analogue.

Effect of the long-acting insulin analogues glargine and degludec on cardiomyocyte cell signalling and function.

BACKGROUND: The effects of insulin on cardiomyocytes, such as positive inotropic action and glucose uptake are well described. However, in vitro studies comparing long-acting insulin analogues with regard to cardiomyocyte signalling and function have not been systematically conducted. METHODS: Insulin receptor (IR) binding was assessed using membrane embedded and solubilised IR preparations. Insulin signalling was analysed in adult rat ventricular myocytes (ARVM) and HL-1 cardiac cells. Inotropic effects were examined in ARVM and the contribution of Akt to this effect was assessed by specific inhibition with triciribine. Furthermore, beating-rate in Cor.4U(®) human cardiomyocytes, glucose uptake in HL-1 cells, and prevention from H2O2 induced caspase 3/7 activation in cardiac cells overexpressing the human insulin receptor (H9c2-E2) were analysed. One-way ANOVA was performed to determine significance between conditions. RESULTS: Insulin degludec showed significant lower IR affinity in membrane embedded IR preparations. In HL-1 cardiomyocytes, stimulation with insulin degludec resulted in a lower Akt(Ser(473)) and Akt(Thr(308)) phosphorylation compared to insulin, insulin glargine and its active metabolite M1 after 5- and 10-min incubation. After 60-min treatment, phosphorylation of Akt was comparable for all insulin analogues. Stimulation of glucose uptake in HL-1 cells was increased by 40-60 %, with a similar result for all analogues. Incubation of electrically paced ARVM resulted for all insulins in a significantly increased sarcomere shortening, contractility- and relaxation-velocity. This positive inotropic effect of all insulins was Akt dependent. Additionally, in Cor.4U(®) cardiomyocytes a 10-20 % increased beating-rate was detected for all insulins, with slower onset of action in cells treated with insulin degludec. H9c2-E2 cells challenged with H2O2 showed a fivefold increase in caspase 3/7 activation, which could be abrogated by all insulins used. CONCLUSIONS: In conclusion, we compared for the first time the signalling and functional impact of the long-acting insulin analogues insulin glargine and insulin degludec in cardiomyocyte cell models. We demonstrated similar efficacy under steady-state conditions relative to regular insulin in functional endpoint experiments. However, it remains to be shown how these results translate to the in vivo situation.

No evidence for αGal epitope transfer from media containing FCS onto human endothelial cells in culture.

BACKGROUND: Current clinical applications of cell therapies and tissue engineered (TE) constructs aim to generate non-immunogenic cells in the best-case scenario of autologous origin. As the cells are cultured, it is theoretically possible that immunoreactive molecules present in xenogenic cell culture media components, such as fetal calf serum (FCS), are transmitted in the culturing process. This problem has propelled the search for xeno-free culture media; however, in vitro culturing of many cell types, especially TE constructs which consist of several cell types, still relies to a great extent on FCS. In this study, we investigated the degree to which xenoantigens are transmitted to human endothelial cells (EC) cultured in medium containing FCS. METHODS: Human EC were isolated from pulmonary artery fragments and atrial appendage tissue samples by enzymatic digestion followed by magnetic-activated cell separation (MACS) utilizing CD31 antibodies. The cells were cultured in EGM-2 medium containing 10% FCS for several passages. Griffonia Simplicifolia Lectin I - Isolectin B4 (GSL I-B4) was used to detect cell surface-bound αGal epitopes either microscopically or flow cytometrically. Antibody binding to cells exposed to human sera prepared from healthy blood donors was investigated to detect surface-located xenoantigens. An antibody-dependent cytotoxicity assay was conducted with heat-inactivated human serum supplemented with rabbit complement and analyzed by flow cytometry after staining for living and dead cells (LIVE/DEAD assay kit). In all experiments, cells cultured in EGM-2 supplemented with 10% human serum (HS) served as controls. RESULTS: Human EC were isolated and cultured successfully for ≥6 passages. GSL I-B4 staining showed no difference between human EC cultured in FCS and in HS. In contrast to porcine EC which showed strong staining with GSL I-B4 and binding of preformed human serum antibodies, human EC cultured in FCS media did not bind human antibodies from high titer anti-αGal and anti-Neu5GC antibody serum. Along these lines, the antibody-dependent cytotoxicity assay showed that human EC cultures independent of FCS or HS usage were not affected, whereas about 40% of porcine EC did not survive. CONCLUSION: Despite culturing cells in an environment containing xenoantigens, we were unable to demonstrate the translocation of xenogenic epitopes onto the surface of human EC or find an increased sensitivity in preformed human xenoantibody-dependent complement activity. Therefore, our results suggest that the use of human cells for TE or cell therapy grown in cell culture systems complemented with FCS does not necessarily lead to an acute rejection reaction upon implantation.

Pharmacological manipulation of blood and lymphatic vascularization in ex vivo-cultured mouse embryos.

Formation of new blood and lymphatic vessels is involved in many physiological and pathological processes, including organ and tumor growth, cancer cell metastasis, fluid drainage and lymphedema. Therefore, the ability to manipulate vascularization in a mammalian system is of particular interest to researchers. Here we describe a method for pharmacological manipulation of de novo and sprouting blood and lymphatic vascular development in ex vivo-cultured mouse embryos. The described protocol can also be used to evaluate the properties of pharmacological agents in growing mammalian tissues and to manipulate other developmental processes. The whole procedure, from embryo isolation to image quantification, takes 3-5 d, depending on the analysis and age of the embryos.

ADME related profiling in 96 and 384 well plate format--a novel and robust HT-assay for the determination of lipophilicity and serum albumin binding.

The failure of about half of the drug candidates is associated with poor pharmacokinetic properties leading to a huge loss of time and money [1]. Early profiling of drug like properties provides important information in order to screen out insoluble, poorly absorbed and toxic compounds. Today, large compound libraries have to be screened, and of course the total number of compounds will rise over the next years leading to a growing demand for fully automated assays. A balance between quality, speed, throughput, cost and information content can be accomplished by the careful selection of assays and experimental conditions. Here we describe a novel 384 well format assay for two important ADME related descriptors (lipophilicity and serum protein binding) as input parameters for a precise prediction of fraction absorbed, blood/organ distribution coefficients and permeability, in order to maximize the information about a compound at an early stage of discovery.

The acetylcholine fiber density of the neocortex is altered by isolated rearing and early methamphetamine intoxication in rodents.

Alterations in the cholinergic physiology of the brain were the first to be observed when research on environmental influences on postnatal brain development began 35 years ago. Since then, the effects of isolated rearing (IR) or early pharmacological insults have been shown not only on the physiology, but also the anatomy of a variety of transmitter systems. The cholinergic fiber density, however, still remained to be assessed. We therefore used a histochemical procedure to stain cholinergic fibers in the brains of young adult gerbils reared either in groups in enriched environments or isolated in standard makrolon cages. Half of the animals from each rearing condition had received a single high dose of methamphetamine on postnatal day 14. Fiber densities were measured by computerized image analysis in the medial and orbital prefrontal cortex (PFC), dysgranular and granular insular cortex, sensorimotor cortices, and the entorhinal cortex of both hemispheres. Isolation rearing increased the cholinergic fiber densities in the prefrontal cortices of the left hemisphere and in the entorhinal cortex of the right hemisphere by about 10%, with no effect in the respective contralateral side. The early methamphetamine intoxication showed no influence in prefrontal and entorhinal cortices, but diminished the acetylcholine (ACh) innervation of the forelimb area of cortex in both hemispheres in IR gerbils and of the left hemisphere in ER gerbils, and reduced the acetylcholine innervation in the hindlimb area in both sides in both rearing groups. These results demonstrate that (a) cholinergic fiber density is differentially regulated in different cortical areas and (b) the plasticity of the cholinergic system can only be understood in the interplay with other neuromodulatory innervations.

Lipophilicity - beyond octanol/water: a short comparison of modern technologies.

Sorting out new chemical entities (NCE) with inappropriate absorption, distribution, metabolism, excretion (ADME) behaviour at an early stage of drug discovery and development is a major challenge in pharmaceutical profiling. An accepted strategy to predict absorption is the measurement of the permeability of a drug candidate. One step earlier the lipophilicity of a compound is determined, which is directly related to permeability and fraction absorbed. Here, we compare today's state of the art technologies for a fast measurement of lipophilicity.: