Plasma Free Levels Research Articles

The Pathogenesis, Treatment and Prevention of Osteoporosis in Men

Testosterone stimulates longitudinal and appositional growth during childhood, whereas estrogen induces epiphysial closure. During adulthood, testosterone continues to stimulate periosteal growth, whereas estrogen is important for the maintenance of trabecular bone mass and structure. In males, testosterone is aromatized to estradiol. Both free and bioavailable plasma levels of testosterone and estradiol decrease with age in males, and fracture risk is associated with low estradiol levels. Testosterone may increase muscle mass and prevent fractures related to falls. Younger hypogonadal males should be treated with testosterone to attain peak bone mass and increase bone mineral density (BMD). Older hypogonadal males should be treated in cases of osteoporosis, reduced muscle strength and increased risk of falling. Secondary hyperparathyroidism caused by calcium and vitamin D insufficiency may reduce bone mass and strength and increase fracture risk and should be avoided. Since calcium supplementation has been associated with an increased risk of cardiovascular complications and renal stones, the dose should be tailored to the habitual daily calcium intake. Lifestyle-related risk factors (smoking, alcohol consumption, lack of physical activity and low body weight) should be addressed. The antifracture efficacy of antiresorptive and anabolic treatment for osteoporosis has not been documented in larger randomized controlled studies. However, changes in BMD and bone markers suggest similar effects in males and females of bisphosphonates (alendronate, risedronate, ibandronate, zoledronic acid), nasal calcitonin, denosumab and teriparatide (parathyroid hormone [1-34]). The antiresorptive drugs should be used in males with BMD T-score less than -2.5 and one or more risk factors, or with hip and vertebral fractures. It seems appropriate to recommend a higher cut-off T-score (e.g. less than -1.0 standard deviation [SD]) in glucocorticoid-induced osteoporosis and in patients receiving androgen deprivation therapy because of the fast initial bone loss. Anabolic treatment should be used in more severe spinal fracture cases, including glucocorticoid-induced osteoporosis.

Comparison of Fluconazole Renal Penetration Levels in Healthy and Candida albicans-Infected Wistar Rats

The aims of this study were to evaluate free levels of fluconazole (FCZ) in the kidneys of healthy and Candida albicans-infected Wistar rats using microdialysis and to establish the relationship between free renal and total plasma levels under both conditions. Microdialysis recovery rates were determined in vitro by dialysis, and retrodialysis recovery rates were determined in vivo by retrodialysis. The recovery rate was around 50%, independent of the method, drug concentration, or condition (in vitro or in vivo) used. FCZ kidney penetration in healthy and infected rats was investigated after the administration of 10 mg/kg of body weight intravenously (i.v.) or 50 mg/kg orally (n = 6/group) and blood and microdialysate sample harvesting at predetermined time points up to 24 and 18 h, respectively. There were no statistical differences between the area under the free concentration-time curve (AUC(0-∞)) values in plasma and in tissue for either healthy or infected groups for the same dose regimen investigated. The antifungal tissue penetrations were similar for both doses and under all conditions investigated (ranging from 0.77 to 0.84). The unbound fraction of FCZ was concentration independent (86.0% ± 2.0%), allowing the prediction of free renal levels using pharmacokinetic parameters obtained from total plasma fitting. The results showed that free renal and free plasma levels are similar in healthy and systemically C. albicans-infected rats. Therefore, free plasma levels are a good surrogate to estimate free FCZ renal concentrations in systemic candidiasis and can be used to optimize dosing regimens for this drug.

Effect of hemodialysis session on the dynamics of carnitine ester profile changes in l-carnitine pretreated end-stage renal disease patients

Carnitine deficiency is common in end-stage renal disease (ESRD) patients receiving hemodialysis (HD) treatment. We investigated the effects of L-carnitine supplementation on acyl carnitine (AC) profile and the changes of distinct ACs during a single HD session in long-term L-carnitine pretreated ESRD patients. Twenty non-diabetic adult patients and 37 healthy controls were studied. Blood samples were drawn before and after 12 weeks of carnitine supplementation, then hourly during an HD session, as well as 30 min after the end of the session. Free and individual AC plasma levels were determined by using ESI MS/MS technique. HD patients showed lower free- and total carnitine levels and elevated ACs and acyl/free carnitine ratio before carnitine supplementation. The L-carnitine supplementation resulted in dramatic elevation of all carnitine esters. The HD session induced a progressive decline in free, short-chain, and dicarboxylic ACs (~80 % of pre-HD amount was washed out); the decrease of medium-chain ACs proved to be more moderate (~60 % washed out), whereas the long-chain ACs remained unaffected. Already 30 min after HD, a substantial increase was seen in free carnitine concentration (reaching 26 % of predialysis level) and the ACs also started to replenish (to 21-52 % of predialysis levels), without further exogenous carnitine load. The washout induced by HD session results in variable depletion of short-, medium-, and long-chain carnitine esters in carnitine-pretreated patients; the recovery of the circulating carnitine esters from the body stores occurs within 30 min after the cessation of the HD procedure.

Skin and soft tissue concentrations of tedizolid (formerly torezolid), a novel oxazolidinone, following a single oral dose in healthy volunteers

Plasma concentrations of antimicrobial drugs have long been used to correlate exposure with effect, yet one cannot always assume that unbound plasma and tissue concentrations are similar. Knowledge about unbound tissue concentrations is important in the development of antimicrobial drugs, since most infections are localised in tissues. Therefore, a clinical microdialysis study was conducted to evaluate the distribution of tedizolid (TR-700), the active moiety of the antimicrobial prodrug tedizolid phosphate (TR-701), into interstitial fluid (ISF) of subcutaneous adipose and skeletal muscle tissues following a single oral 600mg dose of tedizolid phosphate in fasting conditions. Twelve healthy adult subjects were enrolled. Two microdialysis probes were implanted into the thigh of each subject, one into the vastus medialis muscle and one into subcutaneous adipose tissue. Probes were calibrated using retrodialysis. Dialysate samples were collected every 20min for 12h following a single oral dose of 600mg tedizolid phosphate, and blood samples were drawn over 24h. Unbound tedizolid levels in plasma were similar to those in muscle and adipose tissue. The ratios of unbound (free) AUC in tissues over unbound AUC in plasma (fAUCtissue/fAUCplasma) were 1.1±0.2 and 1.2±0.2 for adipose and muscle tissue, respectively. The median half-life was 8.1, 9.2 and 9.6h for plasma, adipose tissue and muscle tissue, respectively. Mean protein binding was 87.2±1.8%. The study drug was very well tolerated. The results of this study show that tedizolid distributes well into ISF of adipose and muscle tissues. Unbound levels of tedizolid in plasma, adipose tissue and muscle tissue were well correlated. Free plasma levels are indicative of unbound levels in the ISF of muscle and adipose tissues.

Pancreatic islets from dexamethasone-treated rats show alterations in global gene expression and mitochondrial pathways

Chronic administration of glucocorticoids (GC) leads to characteristic features of type 2 diabetes in mammals. The main action of dexamethasone in target cells occurs through modulation of gene expression, although the exact mechanisms are still unknown. We therefore investigated the gene expression profile of pancreatic islets from rats treated with dexamethasone using a cDNA array screening analysis. The expression of selected genes and proteins involved in mitochondrial apoptosis was further analyzed by PCR and immunoblotting. Insulin, triglyceride and free fatty acid plasma levels, as well as glucose-induced insulin secretion, were significantly higher in dexamethasone-treated rats compared with controls. Out of 1176 genes, 60 were up-regulated and 28 were down-regulated by dexamethasone treatment. Some of the modulated genes are involved in apoptosis, stress response, and proliferation pathways. RT-PCR confirmed the cDNA array results for 6 selected genes. Bax α protein expression was increased, while Bcl-2 was decreased. In vivo dexamethasone treatment decreased the mitochondrial production of NAD(P)H, and increased ROS production. Concluding, our data indicate that dexamethasone modulates the expression of genes and proteins involved in several pathways of pancreatic-islet cells, and mitochondria dysfunction might be involved in the deleterious effects after long-term GC treatment.

A Genome-Wide Association Study of the Protein C Anticoagulant Pathway

The Protein C anticoagulant pathway regulates blood coagulation by preventing the inadequate formation of thrombi. It has two main plasma components: protein C and protein S. Individuals with protein C or protein S deficiency present a dramatically increased incidence of thromboembolic disorders. Here, we present the results of a genome-wide association study (GWAS) for protein C and protein S plasma levels in a set of extended pedigrees from the Genetic Analysis of Idiopathic Thrombophilia (GAIT) Project. A total number of 397 individuals from 21 families were typed for 307,984 SNPs using the Infinium® 317 k Beadchip (Illumina). Protein C and protein S (free, functional and total) plasma levels were determined with biochemical assays for all participants. Association with phenotypes was investigated through variance component analysis. After correcting for multiple testing, two SNPs for protein C plasma levels (rs867186 and rs8119351) and another two for free protein S plasma levels (rs1413885 and rs1570868) remained significant on a genome-wide level, located in and around the PROCR and the DNAJC6 genomic regions respectively. No SNPs were significantly associated with functional or total protein S plasma levels, although rs1413885 from DNAJC6 showed suggestive association with the functional protein S phenotype, possibly indicating that this locus plays an important role in protein S metabolism. Our results provide evidence that PROCR and DNAJC6 might play a role in protein C and free protein S plasma levels in the population studied, warranting further investigation on the role of these loci in the etiology of venous thromboembolism and other thrombotic diseases.

On the relationship between block of the cardiac Na⁺ channel and drug-induced prolongation of the QRS complex.

Inhibition of the human cardiac Na(+) channel (hNa(v) 1.5) can prolong the QRS complex and has been associated with increased mortality in patients with underlying cardiovascular disease. The safety implications of blocking hNa(v) 1.5 channels suggest the need to test for this activity early in drug discovery in order to design out any potential liability. However, interpretation of hNa(v) 1.5 blocking potency requires knowledge of how hNa(v) 1.5 block translates into prolongation of the QRS complex. We tested Class I anti-arrhythmics, other known QRS prolonging drugs and drugs not reported to prolong the QRS complex. Their block of hNa(v) 1.5 channels (as IC(50) values) was measured in an automated electrophysiology-based assay. These IC(50) values were compared with published reports of the corresponding unbound (free) plasma concentrations attained during clinical use (fC(max)) to provide an IC(50) : fC(max) ratio. KEY RESULTS For 42 Class I anti-arrhythmics and other QRS prolonging drugs, 67% had IC(50) : fC(max) ratios <30. For 55 non-QRS prolonging drugs tested, 72% had ratios >100. Finally, we determined the relationship between the IC(50) value and the free drug concentration associated with prolongation of the QRS complex in humans. For 37 drugs, QRS complex prolongation was observed at free plasma concentrations that were about 15-fold lower than the corresponding IC(50) at hNa(v) 1.5 channels. A margin of 30- to 100-fold between hNa(v) 1.5 IC(50) and fC(max) appears to confer an acceptable degree of safety from QRS prolongation. QRS prolongation occurs on average at free plasma levels 15-fold below the IC(50) at hNa(v) 1.5 channels. This article is commented on by Gintant et al., pp. 254-259 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01433.x.

JTT-130, a novel intestine-specific inhibitor of microsomal triglyceride transfer protein, suppresses high fat diet-induced obesity and glucose intolerance in Sprague-Dawley rats

Microsomal triglyceride transfer protein (MTP) takes part in the mobilization and secretion of triglyceride-rich lipoproteins from enterocytes and hepatocytes. We investigated the effects of JTT-130, a novel intestine-specific MTP inhibitor, on high fat diet-induced obesity and glucose intolerance. Male Sprague-Dawley rats were fed a 3.1% fat diet or a 35% fat diet with or without JTT-130 as a food admixture (0.029%). Food intake, body weight, abdominal fat, hepatic triglyceride, faecal free fatty acids and plasma levels of glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) were assessed. Plasma levels of glucose and insulin were measured during intraperitoneal glucose tolerance tests. In addition, indirect calorimetry was performed on rats fed with a 35% fat diet. JTT-130 treatment decreased body weights, abdominal fat and hepatic triglyceride with suppression of food intake and elevation of faecal free fatty acids and plasma GLP-1 and PYY levels in rats fed with the 35% fat diet, whereas no significant effects on these parameters except for increased faecal free fatty acids were observed in rats fed with the 3.1% fat diet. JTT-130 treatment decreased plasma levels of glucose and insulin during intraperitoneal glucose tolerance tests on rats fed with the 35% fat diet, but not on rats fed with the 3.1% fat diet. JTT-130-treated rats showed increased O(2) consumption and CO(2) production on a 35% fat diet. JTT-130 suppresses high fat diet-induced obesity and glucose intolerance with suppression of food intake and fat absorption and could be useful for prevention and treatment of obesity and obesity-related insulin resistance.

Acipimox during exercise points to an inhibitory feedback of GH on ghrelin secretion in bulimic and healthy women

ObjectiveGhrelin is predominantly produced by the stomach and the growth hormone (GH)-ghrelin feedback loop between the stomach and the pituitary gland has recently been suggested. The disruption of the gut-brain axis might be involved in bulimia nervosa (BN). MethodsWe investigated responses of plasma GH, ghrelin, and neuropeptide Y (NPY) concentrations to exercise or to exercise after the administration of the antilipolytic drug Acipimox (Aci) in seven BN patients and seven healthy women (C). Aci was administered 1h before exercise (45min, 2W/kg of lean body mass/LBM/). Ghrelin, GH, NPY, free fatty acids (FFA) and glycerol plasma levels were measured during the test using commercial kits. ResultsThe exercise induced an increase in plasma GH, NPY and FFA in both groups and a decrease in plasma ghrelin levels only in BN patients. Exercise after Aci administration resulted in an increase in plasma GH, and a decrease in plasma ghrelin in both groups; NPY increased more in BN patients. Exercise-induced FFA increase was depressed after Aci. ConclusionsWe conclude that the Aci-induced suppression in plasma ghrelin levels during exercise in both groups suggests a negative feedback of GH on ghrelin secretion. Observed changes in plasma FFA levels were not related to changes in GH and ghrelin levels.

Increased risk of gestational vascular complications in women with low free tissue factor pathway inhibitor plasma levels out of pregnancy

Tissue factor pathway inhibitor (TFPI) plays a crucial role in haemostasis by regulating TF-induced initiation of coagulation. Since it is expressed by endothelial and trophoblastic cells, TFPI is of particular importance at the placental level and might be involved in the occurrence of gestational vascular complications (GVC). In the present study, we investigated plasma free TFPI antigen in four groups of women: healthy non-pregnant women without history of pregnancy complications; women at the beginning (<12 weeks) and women during the third trimester of a normal pregnancy; women with late pregnancy complications (pre-eclampsia / HELLP syndrome, intra-uterine fetal death, fetal growth retardation) at the time of obstetrical event and/or at distance from pregnancy. In normal pregnancy, TFPI increased between first trimester and delivery (median 5.0 ng/ml vs. 7.1 ng/ml; p<0.0001) but remained lower than in non-pregnant controls (median 8.2 ng/ml; p<0.0001). In patients, when measured concomitantly to the obstetrical event, TFPI showed no difference with normal late pregnancy levels. In contrast, at distance from pregnancy, in the absence of any hormonal influence, TFPI was significantly lower than in non-pregnant controls (median 5.9 vs. 8.2ng/ml, p < 0.0001). After categorisation into quartiles, an inverse dose-effect relationship was demonstrated between TFPI categories recorded apart from pregnancy and GVC risk, with a crude odds ratio of 43.5 (95% confidence interval 8.2-230) for patients with TFPI values in the lowest quartile (< 5.7 ng/ml). In conclusion, low free TFPI at distance from pregnancy appears to be a strong indicator of GVC risk.

Prothrombotic profile in patients with vasospastic or non vasospastic angina and non significant coronary stenosis

BackgroundPatients with vasospastic (VA) or non vasospastic angina (NVA) without significant coronary stenosis have a reduced risk of infarction but is unclear whether or not this may be attributable to a lack of prothrombotic profile - similar to that present in patients with stable coronary artery disease (CAD).MethodsPlasma levels of von Willebrand factor, total and free tissue factor pathway inhibitor, plasminogen activator inhibitor-1, and fibrinogen were analyzed in 15 patients with stable VA and 23 with NVA, all with vasoconstrictive response to acetylcholine although with different severity. Results were compared with those of 20 age-matched controls and 10 patients with CAD.ResultsPlasma levels of von Willebrand factor in patients with VA or NVA were higher than in controls (207 ± 62 and 203 ± 69% vs 121 ± 38%, p < 0.001) and tended to be lower than in CAD patients (264 ± 65, p = 0.145). They also presented higher total tissue factor pathway inhibitor (123 ± 18 and 111 ± 25 vs 88 ± 14, ng/ml p < 0.001) and plasminogen activator inhibitor-1 levels than controls (51 ± 30 and 52 ± 31% vs 19 ± 9 ng/ml, p < 0.001) and similar to CAD patients (134 ± 23 and 62 ± 31, respectively, ns). Moreover, free tissue factor pathway inhibitor plasma levels were lower than controls (18 ± 5 and 17 ± 5 vs 23 ± 8 ng/ml, p = 0.002) and similar to CAD patients (14 ± 5, ns). Despite this prothrombotic condition none of VA or NVA patients presented a myocardial infarction during a 9 year follow-up, an observation also reported in larger series.ConclusionsDuring a stable phase of their disease, patients with VA or NVA present a prothrombotic profile that might eventually contribute to occurrence of myocardial infarction. The rarity of these events, however, may suggests that ill defined factors would protect these patients from coronary plaque rupture/fissure.

Impaired aldosterone responsiveness in corticosteroid binding globulin deficient mice

Corticosteroid binding globulin (CBG) is the high affinity plasma carrier protein for cortisol. It keeps the steroids inactive, prevents them from degradation and defines the amount of free hormone acting on target tissues. Previous findings have shown insufficient responsiveness of corticosterone in peripheral tissues in CBG⁻(/)⁻ mice despite elevated free plasma corticosterone. In the large intestine, glucocorticoids synergistically enhance the pro-absorptive effects of aldosterone. We therefore hypothesized that CBG⁻(/)⁻ mice have reduced responsiveness to aldosterone. We used CBG⁻(/)⁻ and CBG(+/+) mice to investigate distal colonic electrogenic Na(+) absorption. An Ussing chamber was used to quantify amiloride-sensitive Na(+) transport in distal colonic mucosa (ΔI(sc) (amil)) as a measure of the physiological effect of aldosterone. No differences were observed in ΔI(sc) (amil) or aldosterone levels in animals on control diet. When Na(+) restricted, CBG(+/+) mice responded with a marked up-regulation of ΔI(sc) (amil) (25-fold). In CBG⁻(/)⁻ mice this up-regulation was greatly attenuated as seen in a markedly reduced amiloride-sensitive short circuit current (reduced by ∼50%), a reduced ability to lower faecal Na(+) excretion and a significantly attenuated up-regulation of the ENaC channel γ-subunit. Diet-induced increases of total plasma aldosterone were similar in both genotypes, but CBG⁻(/)⁻ mice had an increased free plasma aldosterone fraction. This study defines the functional hyporesponsiveness and aldosterone resistance in distal colon of CBG⁻(/)⁻ mice. This resistance occurs despite sufficient free corticosterone plasma level. Thus, steroid actions require an intrinsic but unknown function of CBG, which allows the sufficient supply of the hormone/s to the target tissue.

Exposure to urban stressors and free testosterone plasma values

The chemical agents present in the environment, such as traffic pollutants, may affect male fertility. Traffic policemen are daily exposed to traffic pollutants. The aim of this study is to evaluate whether occupational exposure to urban stressors could cause alterations in free testosterone plasma values in male traffic policemen versus administrative staff of Municipal Police of a big Italian city. Both groups were divided into two subgroups based on age (first group: 30-40years; second group: 41-50years) to assess whether age could affect laboratory results of free testosterone plasma levels in traffic policemen versus controls. The characterization of exposure to urban pollutants for traffic policemen was assessed using the concentrations of pollutants monitored in fixed stations. A total of 220 subjects were studied: 110 traffic policemen and 110 controls, after excluding subjects with main confounding factors. Mean free testosterone values were significantly lower in traffic policemen than in controls (P<0.001). Such statistical reduction persisted stratifying the mean testosterone values for classes of age (30-40 and 41-50year) of workers (respectively P<0.001 and P<0.001). The distribution into classes of testosterone values in traffic policemen and in controls was significant (P<0.001), and this result persisted after the stratification for classes of age of workers (30-40year: P<0.001) (41-50 year: P<0.001). According to data in literature, free testosterone plasma levels could be used as an early biological marker, to be employed in occupational sets, valuable for the group, even before the onset of values out of range and of fertility disorders.

Interaction with caveolin-1 modulates vascular ATP-sensitive potassium (KATP) channel activity

ATP-sensitive potassium channels (K(ATP) channels) of arterial smooth muscle are important regulators of arterial tone, and hence blood flow, in response to vasoactive transmitters. Recent biochemical and electron microscopic evidence suggests that these channels localise to small vesicular invaginations of the plasma membrane, known as caveolae, and interact with the caveolae-associated protein, caveolin. Here we report that interaction with caveolin functionally regulates the activity of the vascular subtype of K(ATP) channel, Kir6.1/SUR2B. Pinacidil-evoked recombinant whole-cell Kir6.1/SUR2B currents recorded in HEK293 cells stably expressing caveolin-1 (69.6 +/- 8.3 pA pF(1), n = 8) were found to be significantly smaller than currents recorded in caveolin-null cells (179.7 +/- 35.9 pA pF(1), n = 6; P < 0.05) indicating that interaction with caveolin may inhibit channel activity. Inclusion in the pipette-filling solution of a peptide corresponding to the scaffolding domain of caveolin-1 had a similar inhibitory effect on whole-cell Kir6.1/SUR2B currents as co-expression with full-length caveolin-1, while a scrambled version of the same peptide had no effect. Interestingly, intracellular dialysis of vascular smooth muscle cells with the caveolin-1 scaffolding domain peptide (SDP) also caused inhibition of pinacidil-evoked native whole-cell K(ATP) currents, indicating that a significant proportion of vascular K(ATP) channels are susceptible to block by exogenously applied SDP. In cell-attached recordings of Kir6.1/SUR2B single channel activity, the presence of caveolin-1 significantly reduced channel open probability (from 0.05 +/- 0.01 to 0.005 +/- 0.001; P < 0.05) and the amount of time spent in a relatively long-lived open state. These changes in kinetic behaviour can be explained by a caveolin-induced shift in the channel's sensitivity to its physiological regulator MgADP. Our findings thus suggest that interaction with caveolin-1 suppresses vascular-type K(ATP) channel activity. Since caveolin expression is regulated by cellular free cholesterol and plasma levels of low-density lipoprotein (LDL), this interaction may have implications in both the physiological and pathophysiological control of vascular function.

Influence of etoricoxib on anticonvulsant activity of phenytoin and diazepam in experimental seizure models in mice

Our aim was to investigate the effect of etoricoxib on the anticonvulsant activity of phenytoin and diazepam against seizure models in mice. In addition the acute adverse effect of etoricoxib was assessed with a chimney test. The maximal seizure pattern was induced in mice by giving an alternating current of 50 mA for 0.2 s, while chemical seizures were induced by intraperitoneal injection of pentylenetetrazole at its CD97 dose (97% convulsive dose for the clonic phase). Test drug was administered 45 min before the electrical or chemical induction of seizures in combination with conventional antiepileptics. The ability of the test drug to reduce or abolish the extensor phase of maximal electroshock and clonic-type seizures in the chemical induction method was selected as anti-seizure criteria. Concurrent treatment with etoricoxib at an oral dose of 10 mg/kg reduced the anticonvulsant potency of phenytoin. The protective effects of diazepam against pentylenetetrazole-induced convulsions was significantly increased and the mortality rate was reduced by concurrent treatment with etoricoxib (10 mg/kg p.o.) when compared with diazepam groups. No neurotoxic effect was observed with etoricoxib (10 mg/kg p.o.) and it had no impact on motor coordination in the chimney test in mice. Etoricoxib applied at its highest dose (10 mg/kg) significantly enhanced the free plasma levels of diazepam whereas the free plasma levels of phenytoin were significantly reduced. The obtained results suggest that the preferential cyclooxygenase-2 inhibitor etoricoxib significantly reduced the anticonvulsant action of phenytoin and significantly increased the beneficial action of diazepam against maximal electroshock and pentylenetetrazole-induced convulsions in a mouse model.

The Action of a Novel Fluoroquinolone Antibiotic Agent Antofloxacin Hydrochloride on Human-Ether-à-go-go-Related Gene Potassium Channel

The administration of certain fluoroquinolone antibacterials has recently been linked to QT interval prolongation, raising the clinical concerns over the cardiotoxicity of these agents. In this study, the effects of a novel fluoroquinolone, antofloxacin hydrochloride (AX) on human-ether-à-go-go-related gene (HERG) encoding potassium channels and the biophysical mechanisms of drug action were performed with whole-cell patch-clamp technique in transiently transfected HEK293 cells. The administration of AX caused voltage- and time-dependent inhibition of HERG K+ current (I(HERG/MiRP1)) in a concentration-dependent manner but did not markedly modify the properties of channel kinetics, including activation, inactivation, deactivation and recovery from inactivation as well. In comparison with sparfloxacin (SPX), levofloxacin lactate (LVFX), the potency of AX to inhibit HERG tail currents was the least one, with an IC(50) value of 460.37 microM. By contrast, SPX was the most potent compound, displaying an IC(50) value of 2.69 microM whereas LVFX showed modest potency, with an IC(50) value of 43.86 microM, respectively. Taken together, our data suggest that AX only causes a minor reduction of I(HERG/MiRP1) at the estimated free plasma level.

Imatinib plasma levels: correlation with clinical benefit in GIST patients

Imatinib plasma levels: correlation with clinical benefit in GIST patients

The Blood-Brain Barrier and CNS Penetration: a Drug Discovery Point of View

The Blood-Brain Barrier and CNS Penetration: a Drug Discovery Point of View

Testosterone, tissue factor inhibition and vascular aging

Aging is associated with important changes in the cardiovascular system. A key feature is the development of endothelial dysfunction, with the concomitance of enhanced vascular tone, increased production of reactive oxygen species, a fall in fibrinolytic capacity and a rise in vascular and blood procoagulant activity (1). In parallel, the ability to repair vessel injury is impaired by a decreased number and function of circulating progenitor cells. The decline in sex hormone levels with age in both men and women has been thought to contribute to the increased prevalence of endothelial dysfunction, cardiovascular diseases and cardiac-related mortality with age. More specifically, the higher prevalence of ischaemic heart disease in men compared to pre-menopausal age-matched women has suggested that oestrogens, more than testosterone, may exert cardiovascular protection. However, prospective, cross-sectional, and interventional studies have not confirmed these hypotheses (2–4), and have even fuelled the notion of an “oestrogen-androgen paradox” whereby oestrogens may enhance and testosterone may attenuate athero-thrombotic risk (5). In vivo thrombin generation is largely dependent on tissue factor (TF), a fullyfunctional 45 kDa membrane glycoprotein expressed in vascular smooth muscle cells and adventitial fibroblasts (6). A circulating, often encrypted form can be found on white cells, platelets, and shed microparticles (6). Endothelial cells may express TF upon pro-inflammatory cytokine activation (7). At sites of vessel injury/activation, TF may thus come into contact with blood and interact with FVIIa, initiating coagulation (6). Generated thrombin further induces TF expression on the surface of activated platelets, leading to the propagation of coagulation (6). The initiation of coagulation by TF is a regulated process, controlled mainly by tissue-factor pathway inhibitor (TFPI). This 276-amino acid, reversible, serine protease is produced almost entirely by endothelial cells (6). TFPI contains Kunitz domains that mimic the substrate of the target protease: after binding and inhibiting FXa through one Kunitz domain, TFPI subsequently inhibits the TF-FVIIa complex through another domain (6), limiting thrombus formation to the site of TF exposure. In plasma, only 10% of TFPI is in a free active form, the majority being bound to low-density lipoproteins with little inhibitory function (6). Heparin and shear stress upregulate the expression of TFPI (6), whose free plasma levels correlate with age and with markers of endothelial dysfunction (8). Testosterone is produced in men mainly by Leydig cells, with the adrenal cortex contributing minimally (9). Total serum concentrations approximate 20–30 nM in adult men up to 55 years of age, gradually declining thereafter to about 15 nM (9). Levels below 10–12 nM suggest a condition of hypogonadism (9). Circadian variations account for evening nadirs and morning peaks (9). Up to 60–80% of testosterone is tightly bound to the sex hormone-binding globulin (SHBG), with only 1–3% unbound as free fraction and the remainder loosely linked to albumin (9). The bioavailable fraction typically comprises free and albumin-bound testosterone, but experimental studies show that cellular internalisation of SHBG-bound testosterone also occurs (10). Testosterone and its main metabolite, dihydrotestosterone, exert genomic and Correspondence to: Felicita Andreotti, MD, PhD Institute of Cardiology, Gemelli University Hospital Largo Gemelli 8, 00168 Rome, Italy Tel.: +39 0 630154187, Fax: +39 0 63055535 E-mail felicita.andreotti@iol.it Received: November 17, 2009 Accepted: November 17, 2009 Prepublished online: December 1, 2009

Circulating free nitrotyrosine and cognitive decline

To determine if the circulating nitrotyrosine level significantly correlates with parameters measuring cognitive abilities. One-hundred and twelve community-living subjects (ranging in age from 27 to 98 years) were evaluated for cognitive abilities [Mini Mental State Examination (MMSE) score] and circulating free nitrotyrosine plasma level, as well as for several variables that might influence cognitive abilities (age, education) and nitrotyrosine level (body mass index, haematological parameters, cardiovascular and inflammatory indices). In the sub-group of cognitively impaired subjects (score at MMSE < 23.9), but not in that of cognitively not impaired subjects, a significant inverse correlation exists between nitrotyrosine level and MMSE score (r = -0.378; P < 0.02). The finding, if confirmed by longitudinal studies, could play a role in the management of the subjects with Mild Cognitive Impairment, the clinical condition considered as a transitional state between the changes of cognitive ability in normal aging and dementia.