Impact of reduced P-glycoprotein function on digoxin concentrations in patients with dementia.

AIMS: Patients with Alzheimer's disease (AD), the most common form of dementia, have reduced P-glycoprotein (P-gp) function at the blood-brain barrier. However, the effect of AD on P-gp function in peripheral organs, and the impact on medication efficacy and toxicity is unknown. In this study, clinical chart review and physiologically based pharmacokinetic (PBPK) modelling were employed to determine whether disease-associated changes in P-gp could be assessed from clinically measured digoxin concentrations in patients without and with dementia. METHODS: A retrospective chart review was conducted to compare digoxin dose and concentrations between cohorts. A PBPK model was developed to simulate changes in digoxin concentrations at single and multiple 62.5 and 125 µg/d doses due to reduced P-gp function in peripheral organs. RESULTS: Digoxin concentrations were similar between the nondementia (n = 75) and dementia (n = 72) cohorts (mean ± standard deviation; 0.64 ± 0.31 and 0.60 ± 0.34 ng/mL, respectively; -0.06 to 0.15, 95% confidence interval of difference). PBPK simulations showed that reduced P-gp function resulted in a significant increase in digoxin exposure (AUC), but not in Cmax . For example, when a 2-fold reduction in P-gp function was simulated in older people following multiple 125 µg/d digoxin doses, the AUC over the last dosing interval was increased compared to baseline (24.29 ± 3.94 vs 17.04 ± 3.46 ng/mL*h; 4.52 to 9.98); however, Cmax was similar (1.38 ± 0.20 vs 0.99 ± 0.18 ng/mL; -2.33 to 3.13). CONCLUSION: Clinically measured digoxin concentrations were not statistically different in patients with dementia. Based on PBPK simulations, digoxin AUC may need to be evaluated to adequately assess the impact of reduced P-gp function in peripheral organs.

The expansion of GPCR transactivation-dependent signalling to include serine/threonine kinase receptors represents a new cell signalling frontier.

G protein-coupled receptor (GPCR) signalling is mediated through transactivation-independent signalling pathways or the transactivation of protein tyrosine kinase receptors and the recently reported activation of the serine/threonine kinase receptors, most notably the transforming growth factor-ß receptor family. Since the original observation of GPCR transactivation of protein tyrosine kinase receptors, there has been considerable work on the mechanism of transactivation and several pathways are prominent. These pathways include the "triple membrane bypass" pathway and the generation of reactive oxygen species. The recent recognition of GPCR transactivation of serine/threonine kinase receptors enormously broadens the GPCR signalling paradigm. It may be predicted that the transactivation of serine/threonine kinase receptors would have mechanistic similarities with transactivation of tyrosine kinase pathways; however, initial studies suggest that these two transactivation pathways are mechanistically distinct. Important questions are the relative importance of tyrosine and serine/threonine transactivation pathways, the contribution of transactivation to overall GPCR signalling, mechanisms of transactivation and the range of cell types in which this phenomenon occurs. The ultimate significance of transactivation-dependent signalling remains to be defined but it appears to be prominent and if so will represent a new cell signalling frontier.

Atherogenic, fibrotic and glucose utilising actions of glucokinase activators on vascular endothelium and smooth muscle.

BACKGROUND: Pharmaceutical interventions for diabetes aim to control glycaemia and to prevent the development of complications, such as cardiovascular diseases. Some anti-hyperglycaemic drugs have been found to have adverse cardiovascular effects in their own right, limiting their therapeutic role. Glucokinase activity in the pancreas is critical in enhancing insulin release in response to hyperglycaemia. Glucokinase activators (GKAs) are novel agents for diabetes which act by enhancing the formation of glucose-6-phosphate leading to increased insulin production and subsequent suppression of blood glucose. Little, however, is known about the direct effects of GKAs on cardiovascular cells. METHODS: The effect of the GKAs RO28-1675 and Compound A on glucose utilisation in bovine aortic endothelial cells (BAEC) and rat MIN6 was observed by culturing the cells at high and low glucose concentration in the presence and absence of the GKAs and measuring glucose consumption. The effect of RO28-1675 at various concentrations on glucose-dependent signalling in BAEC was observed by measuring Smad2 phosphorylation by Western blotting. The effect of RO28-1675 on TGF-ß stimulated proteoglycan synthesis was measured by 35S-SO4 incorporation and assessment of proteoglycan size by SDS-PAGE. The effects of RO28-1675 on TGF-ß mediated Smad2C phosphorylation in BAEC was observed by measurement of pSmad2C levels. The direct actions of RO28-1675 on vascular reactivity were observed by measuring arteriole tone and lumen diameter. RESULTS: GKAs were demonstrated to increase glucose utilisation in pancreatic but not endothelial cells. Glucose-activated Smad2 phosphorylation was decreased in a dose-dependent fashion in the presence of RO28-1675. No effect of RO28-1675 was observed on TGF-ß stimulated proteoglycan production. RO28-1675 caused a modest dilation in arteriole but not contractile sensitivity. CONCLUSIONS: GKA RO28-1675 did not increase glucose consumption in endothelial cells indicating the absence of glucokinase in those cells. No direct deleterious actions, in terms of atherogenic changes or excessive vasoactive effects were seen on cells or vessels of the cardiovascular system in response to GKAs. If reflected in vivo, these drugs are unlikely to have their use compromised by direct cardiovascular toxicity.

An Investigation of Sodium Fusidate and Recombinant Cytochrome P450 Enzymes Inhibition In-Vitro.

BACKGROUND: Sodium fusidate (fusidic acid) is an antimicrobial agent that is used in the treatment of staphylococcal and streptococcal infections. Several case reports have noted a drug interaction between sodium fusidate and CYP3A4 metabolised statins, leading to statin toxicity. It is unclear whether sodium fusidate has the potential to cause interactions with other cytochrome P450 enzymes. OBJECTIVE: To investigate the effects of sodium fusidate on recombinant cytochrome P450 enzymes (1A2, 2C9, 2C19, 2D6 and 3A4) in-vitro. METHODS: A range of sodium fusidate concentrations (0.1µM, 1µM, 10µM, 100µM, 300µM, 1000µM and 10000µM) were tested to examine its activity on rCYP1A2, rCYP2C9, rCYP2C19, rCYP2D6 and rCYP3A4 using a luminescent assay with a luciferin substrate. RESULTS: Sodium fusidate inhibited all enzymes at tested concentrations which are relevant to those likely to be achieved in clinical practice. Further, sodium fusidate was found to be a time-dependent inhibitor of all the tested isoenzymes, with the exception of rCYP2C9. CONCLUSION: These findings suggest that there is a potential for sodium fusidate to cause drug interactions when used with other agents that are substrates for rCYP1A2, rCYP2C9, rCYP2C19, rCYP2D6 or rCYP3A4. Understanding the basis of this potential drug interaction will assist in safer use of sodium fusidate in clinical practice.

Suramin inhibits PDGF-stimulated receptor phosphorylation, proteoglycan synthesis and glycosaminoglycan hyperelongation in human vascular smooth muscle cells.

OBJECTIVES: Suramin is a polysulfonated naphthylurea with antiparasitic and potential antineoplastic activity. Suramin's pharmacological actions, which have not yet been fully elucidated, include antagonism of the action of platelet-derived growth factor (PDGF) at its receptor. We investigated the effects of suramin on PDGF-stimulated proteoglycan synthesis. METHODS: Human vascular smooth muscle cells (VSMCs) were incubated in the presence and absence of PDGF and suramin with [(3) H]thymidine or (35) SO4 as radiolabels. Mitogenic response was determined by [(3) H]thymidine incorporation. PDGFß receptor phosphorylation was assessed by western blotting. Proteoglycan size and glycosaminoglycan chain synthesis and size were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Alphascreen phosphotyrosine assay kit was used to investigate PDGFß receptor tyrosine kinase inhibition by suramin. KEY FINDINGS: Suramin decreased PDGF-stimulated proliferation, proteoglycan synthesis and GAG chain hyperelongation. Suramin also directly inhibited PDGFß receptor kinase activity as well as PDGFß receptor phosphorylation in intact VSMCs. CONCLUSIONS: These data show that inhibition of PDGFß receptor phosphorylation in intact cells is necessary to define a fully active PDGF antagonist. They also confirm that PDGFß receptor kinase activity is necessary for PDGF-mediated atherogenic changes in proteoglycan synthesis and support efforts to develop PDGFß receptor antagonists as potential anti-atherosclerotic agents.

(S)-[6]-Gingerol inhibits TGF-ß-stimulated biglycan synthesis but not glycosaminoglycan hyperelongation in human vascular smooth muscle cells.

OBJECTIVES: (S)-[6]-Gingerol is under investigation for a variety of therapeutic uses. Transforming growth factor (TGF)-ß stimulates proteoglycan synthesis, leading to increased binding of low-density lipoproteins, which is the initiating step in atherosclerosis. We evaluated the effects of (S)-[6]-gingerol on these TGF-ß-mediated proteoglycan changes to explore its potential as an anti-atherosclerotic agent. METHODS: Purified (S)-[6]-gingerol was assessed for its effects on proteoglycan synthesis by [(35) S]-sulfate incorporation into glycosaminoglycan chains and [(35) S]-Met/Cys incorporation into proteoglycans and total proteins in human vascular smooth muscle cells. Biglycan level was assessed by real-time quantitative polymerase chain reactions and the effects of (S)-[6]-gingerol on TGF-ß signalling by assessment of the phosphorylation of Smads and Akt by western blotting. KEY FINDINGS: (S)-[6]-Gingerol concentration-dependently inhibited TGF-ß-stimulated proteoglycan core protein synthesis, and this was not secondary to inhibition of total protein synthesis. (S)-[6]-Gingerol inhibited biglycan mRNA expression. (S)-[6]-Gingerol did not inhibit TGF-ß-stimulated glycosaminoglycan hyperelongation or phosphorylation of Smad 2, in either the carboxy terminal or linker region, or Akt phosphorylation. CONCLUSIONS: The activity of (S)-[6]-gingerol to inhibit TGF-ß-stimulated biglycan synthesis suggests a potential role for ginger in the prevention of atherosclerosis or other lipid-binding diseases. The signalling studies indicate a novel site of action of (S)-[6]-gingerol in inhibiting TGF-ß responses.