Per Arkhammar

Inhibition of Insulin Secretion as a New Drug Target in the Treatment of Metabolic Disorders

The pattern of insulin release is crucial for regulation of glucose and lipid haemostasis. Deficient insulin release causes hyperglycemia and diabetes, whereas excessive insulin release can give rise to serious metabolic disorders, such as nesidioblastosis (Persistent Hyperinsulinemic Hypoglycemia of Infancy, PHHI) and might also be closely associated with development of type 2 diabetes and obesity. Type 2 diabetes is characterized by fasting hyperinsulinemia, insulin resistance and impaired insulin release, i.e. reduced first phase insulin release and decreased insulin pulse mass. The beta cell function of patients with type 2 diabetes slowly declines and will ultimately result in beta cell failure and increasing degrees of hyperglycemia. Type 2 diabetes, in combination with obesity and cardiovascular disorders, forms the metabolic syndrome. It has been possible to improve beta cell function and viability in preclinical models of type 1 and type 2 diabetes by reducing insulin secretion to induce beta cell rest. Clinical studies have furthermore indicated that inhibitors of insulin release will be of benefit in treatment or prevention of diabetes and obesity. Pancreatic beta cells secrete insulin in response to increased metabolism and by stimulation of different receptors. The energy status of the beta cell controls insulin release via regulation of open probability of the ATP sensitive potassium (K(ATP)) channels to affect membrane potential and the intracellular calcium concentration [Ca(2+)](i). Other membrane bound receptors and ion channels and intracellular targets that modulate [Ca(2+)](i)will affect insulin release. Thus, insulin release is regulated by e.g. somatostatin receptors, GLP-1 receptors, muscarinic receptors, cholecystokinin receptors and adrenergic receptors. Although the relationship between hyperinsulinemia and certain metabolic diseases has been known for decades, only a few inhibitors of insulin release have been characterized in vitro and in vivo. These include the K(ATP) channel openers diazoxide and NN414 and the somatostatin receptor agonist octreotide.

Identification of akt pathway inhibitors using redistribution screening on the FLIPR and the IN cell 3000 analyzer

The PI3-kinase/Akt pathway is an important cell survival pathway that is deregulated in the majority of human cancers. Despite the apparent druggability of several kinases in the pathway, no specific catalytic inhibitors have been reported in the literature. The authors describe the development of a fluorometric imaging plate reader (FLIPR)-based Akt1 translocation assay to discover inhibitors of Akt1 activation. Screening of a diverse chemical library of 45,000 compounds resulted in identification of several classes of Akt1 translocation inhibitors. Using a combination of classical in vitro assays and translocation assays directed at different steps of the Akt pathway, the mechanisms of action of 2 selected chemical classes were further defined. Protein translocation assays emerge as powerful tools for hit identification and characterization. (Journal of Biomolecular Screening 2005:20-29)

A potent diazoxide analogue activating ATP-sensitive K+ channels and inhibiting insulin release

Aims/hypothesis. To characterise the effects of BPDZ 73 (7-chloro-3-isopropylamino-4H-1,2,4-benzothiadiazine 1,1-dioxide), a newly synthesised diazoxide analogue, on insulin secretory cells.¶Methods. Measurements of 86Rb, 45Ca outflow, membrane potential, [Ca2+]i, insulin release in secretory cells as well as measurements of smooth muscle contractile activity and glycaemia were carried out.¶Results. The analogue BPDZ 73 induced a dose-dependent decrease in insulin output. The IC50 value averaged 0.73 ± 0.05 μmol/l. The drug increased the rate of 86Rb (42K substitute) outflow from perifused rat pancreatic islets. This effect was inhibited by glibenclamide, a KATP channel blocker. Measurements of DiBAC4(3) fluorescence further indicated that BPDZ 73 hyperpolarised the insulin secreting cells. It also decreased 45Ca outflow from pancreatic islets perifused throughout in the presence of 16.7 mmol/l glucose and extracellular Ca2+. By contrast, the drug did not affect the increase in 45Ca outflow mediated by K+ depolarisation. In single beta cells, BPDZ 73 inhibited the glucose-induced but not the K+-induced rise in [Ca2+]i. Moreover, in Wistar rats, i. p. injection of BPDZ 73 provoked a considerable increase in blood glucose concentration whereas diazoxide induced a modest rise in glycaemia. Lastly, the vasorelaxant properties of BPDZ 73 were slightly less pronounced than those of diazoxide.¶Conclusion/interpretation. The inhibitory effect of BPDZ 73 on the insulin-releasing process results from the activation of KATP channels with subsequent decrease in Ca2+ inflow and [Ca2+]i. The drug seems to be a KATP channel opener, more potent and more selective than diazoxide for insulin secreting cells. [Diabetologia (2000) 43: 723-732]

Safety update on the use of recombinant activated factor VII in approved indications

This updated safety review summarises the large body of safety data available on the use of recombinant activated factor VII (rFVIIa) in approved indications: haemophilia with inhibitors, congenital factor VII (FVII) deficiency, acquired haemophilia and Glanzmanns thrombasthenia. Accumulated data up to 31 December 2013 from clinical trials as well as post-marketing data (registries, literature reports and spontaneous reports) were included. Overall, rFVIIa has shown a consistently favourable safety profile, with no unexpected safety concerns, in all approved indications. No confirmed cases of neutralising antibodies against rFVIIa have been reported in patients with congenital haemophilia, acquired haemophilia or Glanzmanns thrombasthenia. The favourable safety profile of rFVIIa can be attributed to the recombinant nature of rFVIIa and its localised mechanism of action at the site of vascular injury. Recombinant FVIIa activates factor X directly on the surface of activated platelets, which are present only at the site of injury, meaning that systemic activation of coagulation is avoided and the risk of thrombotic events (TEs) thus reduced. Nonetheless, close monitoring for signs and symptoms of TE is warranted in all patients treated with any pro-haemostatic agent, including rFVIIa, especially the elderly and any other patients with concomitant conditions and/or predisposing risk factors to thrombosis.

Direct determination of selenium in solid biological materials by graphite furnace atomic absorption spectrometry

A graphite furnace atomic absorption spectrometric method is described for the direct determination of selenium in microgram amounts of solid biological materials, using the so-called cup-in-tube technique. Spectral interferences were minimised by using Zeeman-effect background correction and a “normal” heating rate during atomisation. The stabilising efficiencies of nickel and palladium on selenium were investigated by means of radioactive measurements, and only palladium was found to be useful. The accuracy of the method was established by analysing a number of reference materials. Good agreement with certified/recommended values, and an over-all precision of 10% at the 1 µg g–1 level was obtained using standardisation against a graph based on additions to a solid sample. The method was applied to insulin-producing cell pellets, which could be analysed using standardisation against an aqueous standard graph. The detection limit for selenium in a solid material is 0.15 µg g–1 for a typical sample mass of 0.5 mg.

Dihydroxyacetone-induced Oscillations in Cytoplasmic Free Ca2+ and the ATP/ADP Ratio in Pancreatic β-Cells at Substimulatory Glucose

Glucose stimulation of pancreatic β-cells causes oscillatory influx of Ca2+, leading to pulsatile insulin secretion. We have proposed that this is due to oscillations of glycolysis and the ATP/ADP ratio, which modulate the activity of ATP-sensitive K+ channels. We show here that dihydroxyacetone, a secretagogue that feeds into glycolysis below the putative oscillator phosphofructokinase, could cause a single initial peak in cytoplasmic free Ca2+ ([Ca2+]i) but did not by itself cause repeated oscillations in [Ca2+]i in mouse pancreatic β-cells. However, in the presence of a substimulatory concentration of glucose (4 mm), dihydroxyacetone induced [Ca2+]i oscillations. Furthermore, these oscillations correlated with oscillations in the ATP/ADP ratio, as seen previously with glucose stimulation. Insulin secretion in response to dihydroxyacetone was transient in the absence of glucose but was considerably enhanced and somewhat prolonged in the presence of a substimulatory concentration of glucose, in accordance with the enhanced [Ca2+]i response. These results are consistent with the hypothesized role of phosphofructokinase as the generator of the oscillations. Dihydroxyacetone may affect phosphofructokinase by raising the free concentration of fructose 1,6-bisphosphate to a critical level at which it activates the enzyme autocatalytically, thereby inducing the pulses of phosphofructokinase activity that cause the metabolic oscillations.

Functional identification and monitoring of individual α and β cells in cultured mouse islets of Langerhans

The aim of the present study was to evaluate, by use of fluorescence microscopy and immunofluorescence stainings, the use of a fluorescent membrane potential sensitive probe as a means to identify and monitor changes in membrane potential of individual cell types in whole islets of Langerhans over time. Our work supports the use of the fluorescent probe bis-(1,3 dibutylbarbituric acid) trimethine oxonol (diBAC4(3)), in identification of single α and β cells in the periphery of mouse pancreatic islets cultured on extracellular matrix. At a low extracellular glucose concentration (3 mM), heterogeneous staining of the islets was observed. Approximately 97% of the peripheral cells that stained brightly with diBAC4(3) were glucagon positive. Additional diBAC4(3) studies, demonstrated that an increase in glucose concentration from 3 to 10 mM is paralleled by repolarization of α cells and depolarization of β cells. This suggests that reciprocity of glucagon and insulin release upon glucose stimulation is coupled to divergent changes in membrane potential of these cell types and supports the use of diBAC4(3) as a means to detect changes in secretion in both cell types.

Regulation of cytoplasmic free Ca2+ in insulin-secreting cells

The cytoplasmic free Ca2+ concentration([Ca2+]i) has a fundamental role in the β-cell stimulus-secretion coupling and is regulated by a sophisticated interplay between nutrients, hormones and neurotransmitters. Metabolism of glucose and other nutrients leads to ATP generation, closure of ATP-regulated K+-channels, depolarization, opening of voltage-activated L-type Ca2+-channels, increase in [Ca2+]i and insulin release (1,2). Hormones and neurotransmitters affect the β-cell through the activation of receptors coupled to various effector systems, such as the adenylate cyclase (AC) or phospholipase C (PLC) system (2). Upon activation of these systems, cAMP is formed or phosphatidyl inositol 4,5-bisphosphate is hydrolysed, resulting in the formation of inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol (DAG). Whereas InsP3 mobilizes intracellularly bound Ca2+, most probably from the endoplasmic reticulum, DAG activates protein kinase C (PKC) (1–3). Although InsP3 increases [Ca2+]i, there is little effect on insulin release, suggesting that the trisphosphate is not primarily involved as a signal for exocytosis in the β-cell (3). With regard to PKC, the physiological role is more clear and this enzyme is involved as a modulator of multiple steps in the β-cell signal-transduction pathway (1–3).

The use of recombinant activated factor VII in patients with acquired haemophilia

Acquired haemophilia (AH) is a rare, often severe bleeding disorder characterised by autoantibodies to coagulation factor VIII (FVIII). Observational studies offer crucial insight into the disease and its treatment. Recombinant activated factor VII (rFVIIa, eptacog alfa activated) was available on an emergency and compassionate use basis from 1988 to 1999 at sites in Europe and North America. In 1996, rFVIIa was approved in Europe for the treatment of AH; it was licensed for this indication in the United States in 2006. Recombinant activated FVII is approved for first-line treatment of bleeding episodes and prevention of bleeding in surgical/invasive procedures in patients with AH. This review provides an up-to-date summary of the haemostatic efficacy of rFVIIa in patients with AH, from the first emergency and compassionate use programmes, to patient registries and a post-marketing surveillance study. In acute bleeding episodes, rFVIIa provided high and consistent rates of control, and available data showed that acute bleed control rates were higher for first-line rFVIIa versus salvage rFVIIa. In surgical procedures, rFVIIa also provided high rates of control. In patients with AH, rFVIIa has a high rate of haemostatic efficacy in acute and surgical bleeding episodes.

Possible role of an ischemic preconditioning-like response mechanism in KATP channel opener-mediated protection against streptozotocin-induced suppression of rat pancreatic islet function

Potassium channel openers (KCOs) decrease insulin secretion from beta-cells. Some KCOs also protect against damage to beta-cell function and type 1 diabetes in animal models. Previously we have found that the KCO NNC 55-0118 counteracted islet cell dysfunction, and this was associated with a lowering of the mitochondrial membrane potential (Deltapsi). Presently we aimed to explore whether inhibition of insulin secretion per se or rather inhibition of mitochondrial function correlates to counteraction of beta-cell suppression. For this we used two novel KCOs (NNC 55-0321 and NNC 55-0462), which at certain concentrations have different actions regarding insulin secretion and the Deltapsi, with NNC 55-0321 being a potent inhibitor of Deltapsi and NNC 55-0462 being a potent inhibitor of insulin secretion. At 10 microM NNC 55-0321, but not with NNC 55-0462, the islet ATP content and ATP/ADP ratio was acutely decreased. This was accompanied by a complete protection against streptozotocin-induced suppression of islet insulin secretion using the former KCO. In cardiac research KCOs have been used to induce an ischemic preconditioning (IPC) response. In line with an IPC-like mechanism we found that NNC 55-0321 induced an initial free oxygen radical formation, PKC-epsilon isoform activation and a subsequent phosphorylation of the survival promoting factor Akt. Thus, KCOs may elicit mitochondrial events that resemble classical IPC seen in cardiomyocytes, and this could explain the enhanced islet cell function observed. KCOs with this property may be particularly interesting compounds to study as a rescue therapy during acute episodes of beta-cell suppression/destruction.