Intercalation of metformin into montmorillonite.

Metformin hydrochloride is an extensively used antidiabetic drug that according to the results reported here is able to spontaneously intercalate layered silicates like the montmorillonite clay mineral following an ion-exchange mechanism. The adsorption isotherm from water solutions shows a great affinity of metformin towards the clay mineral, which can retain about thrice the exchange capacity of the clay. The adsorbed excess was easily removed by washing with water, leading to an intercalation compound that contains 93 meq of metformin per 100 g of montmorillonite, matching the CEC value of this clay. The intercalated metformin is arranged in the interlayer space as a monolayer of monoprotonated molecules, which remain strongly entrapped within the solid. These new hybrid materials were characterized by elemental chemical analysis, XRD, FTIR, TG-DTA, and NMR. We preliminary evaluated the use of the metformin-montmorillonite intercalation compound as a drug delivery system, determining the liberation kinetics of metformin at diverse pH values that mimic the gastrointestinal tract. Although the release rate was not totally slowed down, the system seems promising in view of further optimization for drug delivery applications.

IL-1ß Inhibition in Cardiovascular Complications Associated to Diabetes Mellitus.

Diabetes mellitus (DM) is a chronic disease that affects nowadays millions of people worldwide. In adults, type 2 diabetes mellitus (T2DM) accounts for the majority of all diagnosed cases of diabetes. The course of the T2DM is characterized by insulin resistance and a progressive loss of ß-cell mass. DM is associated with a number of related complications, among which cardiovascular complications and atherosclerosis are the main cause of morbidity and mortality in patients suffering from the disease. DM is acknowledged as a low-grade chronic inflammatory state characterized by the over-secretion of pro-inflammatory cytokines, including interleukin (IL)-1ß, which reinforce inflammatory signals thus contributing to the development of complications. In this context, the pharmacological approaches to treat diabetes should not only correct hyperglycaemia, but also attenuate inflammation and prevent the development of metabolic and cardiovascular complications. Over the last years, novel biological drugs have been developed to antagonize the pathophysiological actions of IL-1ß. The drugs currently used in clinical practice are anakinra, a recombinant form of the naturally occurring IL-1 receptor antagonist, the soluble decoy receptor rilonacept and the monoclonal antibodies canakinumab and gevokizumab. This review will summarize the main experimental and clinical findings obtained with pharmacological IL-1ß inhibitors in the context of the cardiovascular complications of DM, and discuss the perspectives of IL-1ß inhibitors as novel therapeutic tools for treating these patients.

The Angiotensin-(1-7)/Mas Axis Counteracts Angiotensin II-Dependent and -Independent Pro-inflammatory Signaling in Human Vascular Smooth Muscle Cells.

Background and Aims: Targeting inflammation is nowadays considered as a challenging pharmacological strategy to prevent or delay the development of vascular diseases. Angiotensin-(1-7) is a member of the renin-angiotensin system (RAS) that binds Mas receptors and has gained growing attention in the last years as a regulator of vascular homeostasis. Here, we explored the capacity of Ang-(1-7) to counteract human aortic smooth muscle cell (HASMC) inflammation triggered by RAS-dependent and -independent stimuli, such as Ang II or interleukin (IL)-1ß. Methods and Results: In cultured HASMC, the expression of inducible nitric oxide synthase (iNOS) and the release of nitric oxide were stimulated by both Ang II and IL-1ß, as determined by Western blot and indirect immunofluorescence or the Griess method, respectively. iNOS induction was inhibited by Ang-(1-7) in a concentration-dependent manner. This effect was equally blocked by two different Mas receptor antagonists, A779 and D-Pro7-Ang-(1-7), suggesting the participation of a unique Mas receptor subtype. Using pharmacological inhibitors, the induction of iNOS was proven to rely on the consecutive upstream activation of NADPH oxidase and nuclear factor (NF)-κB. Indeed, Ang-(1-7) markedly inhibited the activation of the NADPH oxidase and subsequently of NF-κB, as determined by lucigenin-derived chemiluminescence and electromobility shift assay, respectively. Conclusion: Ang-(1-7) can act as a counter-regulator of the inflammation of vascular smooth muscle cells triggered by Ang II, but also by other stimuli beyond the RAS. Activating or mimicking the Ang-(1-7)/Mas axis may represent a pharmacological opportunity to attenuate the pro-inflammatory environment that promotes and sustains the development of vascular diseases.

Visfatin as a novel mediator released by inflamed human endothelial cells.

BACKGROUND: Visfatin is a multifaceted adipokine whose circulating levels are enhanced in different metabolic diseases. Extracellular visfatin can exert various deleterious effects on vascular cells, including inflammation and proliferation. Limited evidence exists, however, on the capacity of human vascular cells to synthesize and release visfatin by themselves, under basal or pro-inflammatory conditions. METHODS AND RESULTS: Intracellular visfatin was detected by Western blot in non-stimulated human umbilical vein endothelial cells (HUVEC). However, exposing HUVEC for 18 h to a series of pro-inflammatory stimulus, such as interleukin (IL)-1ß (1 to 10 ng/mL), tumor necrosis factor-α (1 to 10 ng/mL) or angiotensin II (10 pmol/L to 1 µmol/L) markedly enhanced intracellular visfatin content. Using IL-1ß (10 ng/mL; 18 h), it was determined that the increase in intracellular visfatin, which was paralleled by enhanced visfatin mRNA levels, relied on a signalling mechanism involving both nuclear factor-κB and poly (ADP ribose) polymerase-1 activation. Moreover, IL-1ß modified the sub-cellular localization of visfatin; while in non-stimulated HUVEC immunoreactive visfatin predominantly showed an intra-nuclear granular pattern, in IL-1ß-inflamed cells an extra-nuclear filamentous staining, co-localising with F-actin fibers and suggesting a secretory pattern, was mainly found. Indeed, IL-1ß promoted visfatin secretion, as determined by both ELISA and immunocytochemistry. CONCLUSIONS: Human endothelial cells synthesize and release visfatin, particularly in response to inflammation. We suggest that the inflamed endothelium can be a source of visfatin, which arises as a local inflammatory mediator and a potential therapeutic target to interfere with vascular inflammation.

Visfatin impairs endothelium-dependent relaxation in rat and human mesenteric microvessels through nicotinamide phosphoribosyltransferase activity.

Visfatin, also known as extracellular pre-B-cell colony-enhancing factor (PBEF) and nicotinamide phosphoribosyltransferase (Nampt), is an adipocytokine whose circulating levels are enhanced in metabolic disorders, such as type 2 diabetes mellitus and obesity. Circulating visfatin levels have been positively associated with vascular damage and endothelial dysfunction. Here, we investigated the ability of visfatin to directly impair vascular reactivity in mesenteric microvessels from both male Sprague-Dawley rats and patients undergoing non-urgent, non-septic abdominal surgery. The pre-incubation of rat microvessels with visfatin (50 and 100 ng/mL) did not modify the contractile response to noradrenaline (1 pmol/L to 30 µmol/L), as determined using a small vessel myograph. However, visfatin (10 to 100 ng/mL) concentration-dependently impaired the relaxation to acetylcholine (ACh; 100 pmol/L to 3 µmol/L), without interfering with the endothelium-independent relaxation to sodium nitroprusside (1 nmol/L to 3 µmol/L). In both cultured human umbilical vein endothelial cells and rat microvascular preparations, visfatin (50 ng/mL) stimulated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, as determined by lucigenin-derived chemiluminiscence. The relaxation to ACh impaired by visfatin was restored by the NADPH oxidase inhibitor apocynin (10 µmol/L). Additionally, the Nampt inhibitor APO866 (10 mmol/L to 10 µmol/L), but not an insulin receptor-blocking antibody, also prevented the stimulation of NADPH oxidase and the relaxation impairment elicited by visfatin. Accordingly, the product of Nampt activity nicotinamide mononucleotide (100 nmol/L to 1 mmol/L) stimulated endothelial NADPH oxidase activity and concentration-dependently impaired ACh-induced vasorelaxation. In human mesenteric microvessels pre-contracted with 35 mmol/L potassium chloride, the endothelium-dependent vasodilation to bradykinin (1 nmol/L to 3 µmol/L) was equally impaired by visfatin and restored upon co-incubation with APO866. In conclusion, visfatin impairs endothelium-dependent relaxation through a mechanism involving NADPH oxidase stimulation and relying on Nampt enzymatic activity, and therefore arises as a potential new player in the development of endothelial dysfunction.

[Low-dose cinacalcet reduces serum calcium in patients with primary hyperparathyroidism not eligible for surgery]. / Dosis bajas de cinacalcet reducen el calcio sérico en pacientes con hiperparatiroidismo primario no subsidiario de tratamiento quirúrgico.

We present our experience with low-dose cinacalcet to normalize serum calcium in patients with primary hyperparathyroidism (PHPT) not eligible for surgery. We analyzed the impact of this drug on various parameters of calcium-phosphorus metabolism and its tolerability profile. We recruited 17 patients diagnosed with PHPT who had hypercalcemia and also met one or more of the following inclusion criteria: elevated risk for parathyroidectomy, persistent/recurrent PHPT after previous parathyroid surgery or refusal to undergo surgery. The starting dose of cinacalcet was 30 or 60 mg/day, which was adjusted depending on the degree of calcemia reduction and tolerance to the drug. We observed a reduction in serum calcium that was already evident in the first post-treatment test. Appropriate dose adjustment was performed when required and normal serum calcium levels were achieved in most patients, remaining stable during follow-up. Parathyroid hormone was reduced but not normalized in most patients. Calciuria decreased while serum phosphate and alkaline phosphatase levels increased. Cinacalcet tolerance was generally good at the doses used. The most common adverse effects were weakness, dizziness and asthenia, leading to treatment withdrawal in only one patient. We conclude that low-dose cinacalcet reduces serum calcium efficiently, normalizes calcium levels in most patients with PHPT not eligible for surgical treatment and has a good tolerability profile.

Dosis bajas de cinacalcet reducen el calcio sérico en pacientes con hiperparatiroidismo primario no subsidiario de tratamiento quirúrgico / Low-dose cinacalcet reduces serum calcium in patients with primary hyperparathyroidism not eligible for surgery

Presentamos nuestra experiencia con cinacalcet a dosis bajas, en pacientes con hiperparatiroidismo primario (HPTP) no subsidiario de tratamiento quirúrgico con el objetivo principal de normalizar la calcemia. Analizamos el impacto del fármaco sobre diversos parámetros del metabolismo calcio-fósforo y su perfil de tolerancia. Reclutamos un total de 17 pacientes diagnosticados de HPTP que presentaban hipercalcemia y que reunían además alguno de los siguientes criterios de inclusión: riesgo elevado para paratiroidectomía, HPTP persistente/recurrente tras cirugía paratiroidea previa o rechazo del paciente a la intervención quirúrgica. La dosis inicial de cinacalcet fue de 30 o 60mg/día, la cual se ajustó en función del grado de reducción de la calcemia y la tolerancia al fármaco. Observamos una reducción del calcio sérico que ya resultaba evidente en el primer control postratamiento. Tras el ajuste pertinente de dosis cuando fue preciso, se consiguió normalizar la calcemia en una mayoría de los pacientes, la cual se mantuvo estable a lo largo del seguimiento. La PTH se redujo, aunque no se normalizó en la mayor parte de los pacientes. La calciuria descendió mientras que la fosforemia y la fosfatasa alcalina sérica aumentaron. La tolerancia a cinacalcet fue buena en general a las dosis utilizadas. Los efectos secundarios más frecuentes fueron debilidad, mareos y astenia, y solamente en un paciente motivaron la suspensión del tratamiento. Concluimos que cinacalcet a dosis bajas reduce la calcemia de forma eficaz y consigue una normalización de la misma en una mayoría de pacientes con HPTP no subsidiarios de tratamiento quirúrgico con un buen perfil de tolerancia al fármaco (AU)

We present our experience with low-dose cinacalcet to normalize serum calcium in patients with primary hyperparathyroidism (PHPT) not eligible for surgery. We analyzed the impact of this drug on various parameters of calcium-phosphorus metabolism and its tolerability profile We recruited 17 patients diagnosed with PHPT who had hypercalcemia and also met one or more of the following inclusion criteria: elevated risk for parathyroidectomy, persistent/recurrent PHPT after previous parathyroid surgery or refusal to undergo surgery. The starting dose of cinacalcet was 30 or 60mg/day, which was adjusted depending on the degree of calcemia reduction and tolerance to the drug.We observed a reduction in serum calcium that was already evident in the first post-treatment test. Appropriate dose adjustment was performed when required and normal serum calcium levels were achieved in most patients, remaining stable during follow-up. Parathyroid hormone was reduced but not normalized in most patients. Calciuria decreased while serum phosphate and alkaline phosphatase levels increased. Cinacalcet tolerance was generally good at the doses used. The most common adverse effects were weakness, dizziness and asthenia, leading to treatment withdrawal in only one patient. We conclude that low-dose cinacalcet reduces serum calcium efficiently, normalizes calcium levels in most patients with PHPT not eligible for surgical treatment and has a good tolerability profile (AU)

Visfatin/PBEF/Nampt: A New Cardiovascular Target?

In the last years, a growing interest has emerged toward understanding the role of adipocytokines in the development of cardio-metabolic complications. Five years ago, visfatin/PBEF/Nampt was identified as a novel adipocytokine. In the context of metabolic disorders, extracellular visfatin/PBEF/Nampt was initially claimed as a potentially beneficial molecule due to its insulin-mimetic and glucose-lowering properties. Nevertheless, growing evidence has since then unveiled that visfatin/PBEF/Nampt may rather be a biomarker of inflammation and endothelial damage, and also a direct regulator of the cardiovascular system that modulates cell proliferation and survival, extracellular matrix, vascular reactivity, and inflammation. On one side, the blockade of the deleterious cardiovascular actions of visfatin/PBEF/Nampt is being regarded as a potential approach to prevent and treat, not only cardio-metabolic complications, but also other pathologies implying excessive angiogenesis. Conversely, the administration of visfatin/PBEF/Nampt has shown beneficial effects in different ischemic conditions. Further research is required to evaluate the real value of visfatin/PBEF/Nampt as a pharmacological target.

Conjugated linoleic acid (CLA) and obesity.

BACKGROUND: The term conjugated linoleic acid (CLA) refers to several positional and geometric conjugated dienoic isomers of linoleic acid (LA), of which the trans-10,cis-12 isomer has been reported to reduce adiposity and increase lean mass in mice and other animals when included at

On how insulin may influence ageing and become atherogenic throughout the insulin-like growth factor-1 receptor pathway: in vitro studies with human vascular smooth muscle cells.

BACKGROUND: It is known that growth factors play a role in ageing and atherogenesis, and insulin develops mitogenic activity in vitro. OBJECTIVES: This study focuses on the pathway by which insulin induces proliferation and mobility in vascular smooth muscle cells (SMCs) compared with that of insulin-like growth factor-1 (IGF-1), because they are two basic phenomena for atherogenesis that could also help to understand the role of insulin in the ageing process. METHODS: Bromodeoxyuridine DNA incorporation, chemotaxis and the appearance of membrane ruffles were measured in cultured SMCs after incubation with insulin or IGF-1 in the presence of insulin or IGF-1 receptor-blocking antibodies. RESULTS: Insulin-induced SMC proliferation through the IGF-1 receptors; indeed, the blockade of insulin receptors does not inhibit the mitogenic influence of insulin. On the contrary, insulin-induced cell migration was inhibited by blocking the insulin receptor but not the IGF-1 receptor. Nevertheless, in less differentiated SMCs from non-confluent cultures, the migratory response was significantly higher and insulin lost its receptor specificity. It was stimulated through receptors both for insulin and IGF-1. In these cases the IGF-1 action was similar. Insulin-induced F-actin rearrangements took place through both types of receptors, but IGF-1 was a little more specific through its own receptors. CONCLUSION: The pathway activated by insulin to induce SMC proliferation is not different from that of IGF-1, whereas the unspecific mechanism inducing mobility in growing cells seems to be related to a higher sensitivity response. Cells with the highest mitotic activity have the highest mobility in which stimulation of receptor specificity is lost for either insulin or IGF-1. Extrapolating these results to in vivo, insulin could become relevant for inducing stabilization and also side effects in ageing.

Age-dependent decline of in vitro migration (basal and stimulated by IGF-1 or insulin) of human vascular smooth muscle cells.

Since biological aging causes a decrease in functions such as cell proliferation, we have studied the possible effect of age on the migration capacity of human vascular smooth muscle cells (SMCs). To this aim, the migration activity of cultured SMCs from arteries of male human donors ranging in age from 43-77 years was determined in a Boyden chamber, under basal conditions and after insulin-like growth factor-1 (IGF-1) or insulin stimulation. Migration activity decreased with donor age (r2 = 87%, 85%, and 78%, respectively). IGF-1 and insulin significantly reduced the age-dependent relationship observed in basal conditions, so that, comparing young with old, both IGF-1 and insulin stimulated SMC migration similarly, although the effect of age remained in absolute terms. In this article, we conclude that the age-dependent decline of migration activity--similar to what has already been shown for SMC proliferation--may be part of the biological ageing phenotype, which is not overcome by hormone stimulation.