Preclinical Assessment of the Bioactivity of the Anticancer Coumarin OT48 by Spheroids, Colony Formation Assays, and Zebrafish Xenografts.

In vitro and in vivo pre-clinical screening of novel therapeutic agents are an essential tool in cancer drug discovery. Although human cancer cell lines respond to therapeutic compounds in 2D (dimensional) monolayer cell cultures, 3D culture systems were developed to understand the efficacy of drugs in more physiologically relevant models. In recent years, a paradigm shift was observed in pre-clinical research to validate the potency of new molecules in 3D culture systems, more precisely mimicking the tumor microenvironment. These systems characterize the disease state in a more physiologically relevant manner and help to gain better mechanistic insight and understanding of the pharmacological potency of a given molecule. Moreover, with the current trend in improving in vivo cancer models, zebrafish has emerged as an important vertebrate model to assess in vivo tumor formation and study the effect of therapeutic agents. Here, we investigated the therapeutic efficacy of hydroxycoumarin OT48 alone or in combination with BH3 mimetics in lung cancer cell line A549 by using three different 3D culture systems including colony formation assays (CFA), spheroid formation assay (SFA) and in vivo zebrafish xenografts.

Discovery and assessment of water soluble coumarins as inhibitors of the coagulation contact pathway.

Over the last decade, the coagulation factor XIIa (FXIIa) has seen renewed interest as a therapeutic target. Indeed, its inhibition could offer a protection against thrombosis without increasing the risk of bleeding. Moreover, it could answer the need for a safe prevention of blood-contacting medical devices-related thrombosis. Among the FXII and FXIIa inhibitors already described in literature, organic small-molecular-weight inhibitors are rather left behind. In this study, we were focused on the discovery and assessment of water soluble small molecules. First, a search within our library of compounds flagged two promising hits. Indeed, enzymes and plasma assays suggested they have a greater activity on the contact factors (FXIa, plasma kallikrein and FXIIa) than on the TF pathway. Then, simple pharmacomodulations were undertaken with the aim to design more selective FXIIa inhibitors. This afforded compounds having different degrees of selectivity. All compounds were finally screened in whole blood using an 8-channel microfluidic model and thromboelastometry measurements. Interestingly, all molecules interfered with the thrombus formation and one of them could be considered as a small organic contact inhibitor.

Cost effectiveness of new oral anticoagulants for stroke prevention in patients with atrial fibrillation in two different European healthcare settings.

OBJECTIVES: Our objectives were to investigate the cost effectiveness of apixaban, rivaroxaban, and dabigatran compared with coumarin derivatives for stroke prevention in patients with atrial fibrillation in a country with specialized anticoagulation clinics (the Netherlands) and in a country without these clinics (the UK). METHODS: A decision-analytic Markov model was used to analyse the cost effectiveness of apixaban, rivaroxaban, and dabigatran compared with coumarin derivatives in the Netherlands and the UK over a lifetime horizon. RESULTS: In the Netherlands, the use of rivaroxaban, apixaban, or dabigatran increased health by 0.166, 0.365, and 0.374 quality-adjusted life-years (QALYs) compared with coumarin derivatives, but also increased costs by 5,681, 4,754, and 5,465, respectively. The incremental cost-effectiveness ratios (ICERs) were 34,248, 13,024, and 14,626 per QALY gained. In the UK, health was increased by 0.302, 0.455, and 0.461 QALYs, and the incremental costs were similar for all three new oral anticoagulants (5,118-5,217). The ICERs varied from 11,172 to 16,949 per QALY gained. In the Netherlands, apixaban had the highest chance (37 %) of being cost effective at a threshold of 20,000; in the UK, this chance was 41 % for dabigatran. The quality of care, reflected in time in therapeutic range, had an important influence on the ICER. CONCLUSIONS: Apixaban, rivaroxaban, and dabigatran are cost-effective alternatives to coumarin derivatives in the UK, while in the Netherlands, only apixaban and dabigatran could be considered cost effective. The cost effectiveness of the new oral anticoagulants is largely dependent on the setting and quality of local anticoagulant care facilities.

Meranzin hydrate exhibits anti-depressive and prokinetic-like effects through regulation of the shared α2-adrenoceptor in the brain-gut axis of rats in the forced swimming test.

BACKGROUND: In recent years, the brain-gut axis theory has received increasing attention in studies of depression. However, most studies separately address potential antidepressant and prokinetic treatments. Investigations of drugs that could potentially treat comorbid depression and gastrointestinal (GI) dysfunction via a common mechanism of action have not yet been performed in detail. AIM: To find a common mechanism of action of our patented drug, meranzin hydrate (MH), in the antidepressant and prokinetic treatment. METHODS: The forced swimming test (FST) model of depression, plasma ghrelin measurement, and in vivo and in vitro measurements of GI motility were used. RESULTS: 1. Administration of MH (9 mg/kg) decreased the immobility time during the FST after acute treatment; this effect was inhibited by the alpha 2-adrenoceptor antagonist, yohimbine, but not by the alpha 1-adrenoceptor antagonist, prazosin. 2. After chronic treatment, the immobility time of rats during the FST was decreased significantly by MH (2.25 mg/kg). 3. MH (9 mg/kg) increased plasma ghrelin levels in rats subjected to the FST; this increase was enhanced by the ghrelin receptor agonist, GHRP-6. 4. MH (9 mg/kg) also promoted gastric emptying and intestinal transit in rats with or without FST. 5. In vitro, MH (10 µM) increased jejunal contractions in rats subjected to the FST; this effect was inhibited by yohimbine. Furthermore, the inhibitory effect of yohimbine was partly reversed by the ghrelin receptor agonist, GHRP-6. CONCLUSION: Our study revealed that MH from natural resources exhibits antidepressive and prokinetic-like effects through the regulation of the common mediator, the alpha 2-adrenoceptor.

Toxicology and risk assessment of coumarin: focus on human data.

Coumarin is a secondary phytochemical with hepatotoxic and carcinogenic properties. For the carcinogenic effect, a genotoxic mechanism was considered possible, but was discounted by the European Food Safety Authority in 2004 based on new evidence. This allowed the derivation of a tolerable daily intake (TDI) for the first time, and a value of 0.1 mg/kg body weight was arrived at based on animal hepatotoxicity data. However, clinical data on hepatotoxicity from patients treated with coumarin as medicinal drug is also available. This data revealed a subgroup of the human population being more susceptible for the hepatotoxic effect than the animal species investigated. The cause of the high susceptibility is currently unknown; possible mechanisms are discussed. Using the human data, a TDI of 0.1 mg/kg body weight was derived, confirming that of the European Food Safety Authority. Nutritional exposure may be considerably, and is mainly due to use of cassia cinnamon, which is a popular spice especially, used for cookies and sweet dishes. To estimate exposure to coumarin during the Christmas season in Germany, a telephone survey was performed with more than 1000 randomly selected persons. Heavy consumers of cassia cinnamon may reach a daily coumarin intake corresponding to the TDI.

A multicentre assessment of the endogenous thrombin potential using a continuous monitoring amidolytic technique.

This study assessed the inter-laboratory imprecision associated with the measurement of the endogenous thrombin potential (ETP). The initial studies used techniques that had evolved in each of the participating laboratories. Samples from normal healthy subjects (n = 10), two patients receiving coumarin therapy [International Normalized Ratio approximately 2.0 and approximately 4.0] and a further two subjects receiving treatment with unfractionated heparin (anti-Xa 0.07 i.u./ml and 0.31 i.u./ml) were assayed relative to a lyophilized normal plasma that had arbitrarily been assigned a potency of 100%. Considerable variation in potency estimates was observed between the centres, although individual laboratories using fully automated techniques achieved acceptable levels of imprecision as assessed by the coefficient of variation (CV) (intra-assay CV < 9.5%, inter-assay CV < 12.5%). A second study to assess a similar range of samples, using a standardized assay protocol and incorporating appraisal of two chromogenic substrates, CBS.0068 or Pefachrom TG, demonstrated markedly improved agreement in potency estimates between centres and good correlation (r > 0.96) between the chromogenic substrates. Our data demonstrates that an automated ETP method can be standardized between laboratories and suitable levels of imprecision achieved, using different analysers (COBAS Mira at two centres and an ACL-300R) and two thrombin substrates. This indicates that more widespread use of ETP measurements in clinical laboratories is feasible.

Is treatment of atrial fibrillation in primary care based on thromboembolic risk assessment?

BACKGROUND: Antithrombotic treatment in atrial fibrillation should be guided by the risk of thromboembolic events. Although practice studies have shown underutilization of antithrombotics, it is not clear whether physicians make use of thromboembolic risk stratification in their treatment decisions, as recommended in current guidelines. OBJECTIVE: The aim of the present study was to assess which clinical determinants influence coumarin and aspirin prescription in patients with atrial fibrillation in primary care. METHODS: In a cross-sectional study of 15 computerized general practices covering 38 000 patients in The Netherlands, we identified patients with atrial fibrillation using several search algorithms. Determinants of antithrombotic therapy were assessed using polytomous logistic regression analysis. RESULTS: A total of 247 patients with atrial fibrillation were identified among 10 335 patients aged 55 years or over (prevalence 2.4%). Mean age was 77 years (range 55-95); 51% were male. Overall, 52% of the patients used coumarin and 27% used aspirin. About 50% of patients were also treated by a cardiologist. Referred patients were prescribed coumarin more often (63% versus 35%, P < 0.001). Prior stroke/transient ischaemic attack or systemic embolism [adjusted odds ratio (OR) 5.3, 95% confidence interval (CI) 1.1-24.8], and chronic heart failure (adjusted OR 2.1, 95% CI 0.8-5.1) were independent determinants for coumarin prescription. These associations were less strong for aspirin prescription. However, other established risk factors for future thromboembolic events, such as hypertension and diabetes mellitus, did not lead to higher prescription rates. In contrast, several factors not associated with thromboembolic events, such as chronic (versus paroxysmal) atrial fibrillation (adjusted OR 3.7, 95% CI 1.7-8.2) and longstanding (versus recent) diagnosis (adjusted OR 2.2, 95% CI 1.1-4.5), were also associated with higher coumarin prescription. CONCLUSION: These results illustrate that physicians' decisions about initiating antithrombotic therapy in atrial fibrillation are not evidence based. They are guided partly by thromboembolic risk stratification, but also by factors insignificant for thromboembolic risk.

A benefit-risk assessment of agents used in the secondary prevention of stroke.

Stroke is a major cause of morbidity and mortality. Full assessment of stroke or transient ischaemic attack (TIA) patients is required to identify all risk factors and apply appropriate secondary preventative strategies. Antiplatelet therapies are effective in the secondary prevention of ischaemic stroke and can be justified despite adverse effects such as gastrointestinal haemorrhage. Aspirin (acetylsalicylic acid), aspirin plus dipyridamole, ticlopidine and clopidogrel are all of value but their adverse effect profiles vary significantly. Combinations of antiplatelet agents may offer additional benefit but not all combinations have been studied in stroke patients. Anticoagulation with agents such as warfarin is effective with coexisting atrial fibrillation and other conditions predisposing to cardioembolic stroke. Antihypertensive agents have been extensively studied in the primary prevention of stroke; however, relatively few trials of antihypertensive agents in the secondary prevention of stroke are available. The incidence of adverse effects of antihypertensive agents is relatively low and the benefit-risk profile would tend to favour their use in the secondary prevention of stroke. Recent studies of ACE inhibitors have identified an important role for these agents in the secondary prevention of stroke even in those who are normotensive and in those who have had a haemorrhagic stroke. The incidence of serious adverse effects with ACE inhibitors appears relatively low. Lipid-lowering agents may have a role to play in certain groups of patients with stroke. The incidence of adverse effects is relatively low with HMG-CoA reductase inhibitors. Cigarette smoking is an important risk factor for stroke and evidence is available that smoking cessation does reduce the individual's risk of stroke. Pharmacological agents are available to help smoking cessation. In patients with diabetes mellitus, intensive regimens with insulin and oral hypoglycaemic agents have so far not definitively been shown to reduce the incidence of macrovascular complications such as stroke. Tight glycaemic control has been shown to improve microvascular complications such as retinopathy, nephropathy and neuropathy and hence this is reason enough to advocate the use of these agents. Future developments in the treatment of diabetes may help. Secondary prevention of stroke has improved greatly over the past decade and hopefully will continue to improve. The use of pharmacological agents available currently and in the future will be clarified and refined as further clinical trials report.

Costs and effectiveness of using coumarins before, during and after coronary angioplasty.

BACKGROUND: In the Balloon Angioplasty and Anticoagulation Study (BAAS), coumarins added to routine aspirin therapy before coronary angioplasty reduced cardiac events at the cost of a slightly higher risk of bleeding complications. OBJECTIVE: To determine the cost effectiveness of coumarin treatment, based on the occurrence of both cardiac and bleeding events. METHODS: Effectiveness was measured, applying two definitions, in terms of the number of events occurring at one year. In the first definition, the occurrence of death, myocardial infarction (MI), or stroke was assessed. The second definition also included revascularisations and major bleeding episodes as an event. Costs were limited to direct medical costs. Cost effectiveness was addressed by probability ellipses representing the point estimates and uncertainties surrounding both costs and effectiveness. RESULTS: At 1 year, death, MI or stroke occurred 1.1% less often when treating with aspirin plus coumarins rather than aspirin therapy alone. When revascularisations and major bleeding events were also included, the difference was 5.0%. Overall, the additional costs in relation to coumarin treatment were compensated by a reduction in repeat interventions. When including all costs, the savings associated with coumarin treatment were estimated at Euros 235 per patient after 1 year. The probability that coumarins are cost saving was estimated at 0.85. The probability that coumarins combine additional effectiveness with cost savings was estimated at 0.70 when survival free of MI or stroke as an effectiveness measure was considered, and at 0.83 when survival free of MI, stroke, revascularisation or major bleeding was considered. CONCLUSION: Coumarin therapy added to routine aspirin therapy before coronary angioplasty, and continued during follow-up, may not only be considered more effective but also cost saving relative to aspirin therapy alone.