Lowering grain boundary resistance of BaZr0.8Y0.2O3−δ with LiNO3 sintering-aid improves proton conductivity for fuel cell operation

A novel sintering additive based on LiNO3 was used to overcome the drawbacks of poor sinterability and low grain boundary conductivity in BaZr0.8Y0.2O3-δ (BZY20) protonic conductors. The Li-additive totally evaporated during the sintering process at 1600°C for 6 h, which led to highly dense BZY20 pellets (96.5% of the theoretical value). The proton conductivity values of BZY20 with Li sintering-aid were significantly larger than the values reported for BZY sintered with other metal oxides, due to the fast proton transport in the "clean" grain boundaries and grain interior. The total conductivity of BZY20-Li in wet Ar was 4.45 × 10-3 S cm-1 at 600°C. Based on the improved sinterability, anode-supported fuel cells with 25 μm-thick BZY20-Li electrolyte membranes were fabricated by a co-firing technique. The peak power density obtained at 700°C for a BZY-Ni/BZY20-Li/La0.6Sr0.4Co0.2Fe 0.8O3-δ (LSCF)-BZY cell was 53 mW cm-2, which is significantly larger than the values reported for fuel cells using electrolytes made of BZY sintered with the addition of ZnO and CuO, confirming the advantage of using Li as a sintering aid.

Biotransformation of cholesterol to diosgenin by freely suspended and immobilised cells of Dioscorea bulbifera L.

The cell suspension cultures, established from the friable callus which was risen from the nodal segments of Dioscorea bulbifera L. in Murashige-Skoog (MS) medium supplemented with indole-3-butryic acid (20 mg L- 1), was examined for cell growth in MS medium fed with cholesterol (50 mg L- 1 and 100 mg L- 1) after 8, 10, 12, 14, 16, and 18 days of culture. The growth index of the cell suspension culture on the 8th day was 1.2 and gradually inclined to 1.9 on the 16th day and remained the same at the 18th day. There is no marked difference in the cell growth of cholesterol-treated and control cell suspension culture. The maximum accumulation of diosgenin was noticed on the 14th day in control and cholesterol-treated cell suspension culture and immobilised cell cultures. The highest concentration of diosgenin, 2.94% and 2.14% dry weight, was obtained in immobilised cell culture and cell suspension culture treated with 100 mg L- 1 cholesterol, respectively.

Prostate cancer organoids: A potential new tool for testing drug sensitivity

Recent technical advances have enabled for the first time, reliable in vitro culture of prostate cancer samples as prostate cancer organoids. This breakthrough provides the significant possibility of high throughput drug screening covering the spectrum of prostate cancer phenotypes seen clinically. These advances will enable precision medicine to become a reality, allowing patient samples to be screened for effective therapeutics ex vivo, with tailoring of treatments specific to that individual. This will hopefully lead to enhanced clinical outcomes, avoid morbidity due to ineffective therapies and improve the quality of life in men with advanced prostate cancer.

Pharmacokinetic interactions affecting the antidiabetic repaglinide

Introduction Repaglinide is a short-acting drug, used to reduce postprandial hyperglycaemia in type 2 diabetic patients. Repaglinide is extensively metabolised, and its oral bioavailability is about 60%; its metabolites are mainly excreted into bile. In previous studies, the cytochrome P450 (CYP) 3A4 inhibitors itraconazole and clarithromycin have moderately increased the area under the concentration-time curve (AUC) of repaglinide. Gemfibrozil, a CYP2C8 inhibitor, has greatly increased repaglinide AUC, enhancing and prolonging its blood glucose-lowering effect. Rifampicin has decreased the AUC and effects of repaglinide. Aims The aims of this work were to investigate the contribution of CYP2C8 and CYP3A4 to the metabolism of repaglinide, and to study other potential drug interactions affecting the pharmacokinetics of repaglinide, and the mechanisms of observed interactions. Methods The metabolism of repaglinide was studied in vitro using recombinant human CYP enzymes and pooled human liver microsomes (HLM). The effect of trimethoprim, cyclosporine, bezafibrate, fenofibrate, gemfibrozil, and rifampicin on the metabolism of repaglinide, and the effect of fibrates and rifampicin on the activity of CYP2C8 and CYP3A4 were investigated in vitro. Randomised, placebo-controlled cross-over studies were carried out in healthy human volunteers to investigate the effect of bezafibrate, fenofibrate, trimethoprim, cyclosporine, telithromycin, montelukast and pioglitazone on the pharmacokinetics and pharmacodynamics of repaglinide. Pretreatment with clinically relevant doses of the study drug or placebo was followed by a single dose of repaglinide, after which blood and urine samples were collected to determine pharmacokinetic and pharmacodynamic parameters. Results In vitro, the contribution of CYP2C8 was similar to that of CYP3A4 in the metabolism of repaglinide (< 2 μM). Bezafibrate, fenofibrate, gemfibrozil, and rifampicin moderately inhibited CYP2C8 and repaglinide metabolism, but only rifampicin inhibited CYP3A4 in vitro. Bezafibrate, fenofibrate, montelukast, and pioglitazone had no effect on the pharmacokinetics and pharmacodynamics of repaglinide in vivo. The CYP2C8 inhibitor trimethoprim inhibited repaglinide metabolism by HLM in vitro and increased repaglinide AUC by 61% in vivo (P < .001). The CYP3A4 inhibitor telithromycin increased repaglinide AUC 1.8-fold (P < .001) and enhanced its blood glucose-lowering effect in vivo. Cyclosporine inhibited the CYP3A4-mediated (but not CYP2C8-mediated) metabolism of repaglinide in vitro and increased repaglinide AUC 2.4-fold in vivo (P < .001). The effect of cyclosporine on repaglinide AUC in vivo correlated with the SLCO1B1 (encoding organic anion transporting polypeptide 1, OATP1B1) genotype. Conclusions The relative contributions of CYP2C8 and CYP3A4 to the metabolism of repaglinide are similar in vitro, when therapeutic repaglinide concentrations are used. In vivo, repaglinide AUC was considerably increased by inhibition of both CYP2C8 (by trimethoprim) and CYP3A4 (by telithromycin). Cyclosporine raised repaglinide AUC even higher, probably by inhibiting the CYP3A4-mediated biotransformation and OATP1B1-mediated hepatic uptake of repaglinide. Bezafibrate, fenofibrate, montelukast, and pioglitazone had no effect on the pharmacokinetics of repaglinide, suggesting that they do not significantly inhibit CYP2C8 or CYP3A4 in vivo. Coadministration of drugs that inhibit CYP2C8, CYP3A4 or OATP1B1 may increase the plasma concentrations and blood glucose-lowering effect of repaglinide, requiring closer monitoring of blood glucose concentrations to avoid hypoglycaemia, and adjustment of repaglinide dosage as necessary.

Common and rare variants of the renin-angiotensin system and their relation to antihypertensive drug responses

Most of the diseases affecting public health, like hypertension, are multifactorial by etiology. Hypertension is influenced by genetic, life style and environmental factors. Estimation of the influence of genes to the risk of essential hypertension varies from 30 to 50%. It is plausible that in most of the cases susceptibility to hypertension is determined by the action of more than one gene. Although the exact molecular mechanism underlying essential hypertension remains obscure, several monogenic forms of hypertension have been identified. Since common genetic variations may predict, not only to susceptibility to hypertension, but also response to antihypertensive drug therapy, pharmacogenetic approaches may provide useful markers in finding relations between candidate genes and phenotypes of hypertension. The aim of this study was to identify genetic mutations and polymorphisms contributing to human hypertension, and examine their relationships to intermediate phenotypes of hypertension, such as blood pressure (BP) responses to antihypertensive drugs or biochemical laboratory values. Two groups of patients were investigated in the present study. The first group was collected from the database of patients investigated in the Hypertension Outpatient Ward, Helsinki University Central Hospital, and consisted of 399 subjects considered to have essential hypertension. Frequncies of the mutant or variant alleles were compared with those in two reference groups, healthy blood donors (n = 301) and normotensive males (n = 175). The second group of subjects with hypertension was collected prospectively. The study subjects (n=313) underwent a protocol lasting eight months, including four one-month drug treatment periods with antihypertensive medications (thiazide diuretic, β-blocker, calcium channel antagonist, and an angiotensin II receptor antagonist). BP responses and laboratory values were related to polymorphims of several candidate genes of the renin-angiotensin system (RAS). In addition, two patients with typical features of Liddle’s syndrome were screened for mutations in kidney epithelial sodium channel (ENaC) subunits. Two novel mutations causing Liddle’s syndrome were identified. The first mutation identified located in the beta-subunit of ENaC and the second mutation found located in the gamma-subunit, constituting the first identified Liddle mutation locating in the extracellular domain. This mutation showed 2-fold increase in channel activity in vitro. Three gene variants, of which two are novel, were identified in ENaC subunits. The prevalence of the variants was three times higher in hypertensive patients (9%) than in reference groups (3%). The variant carriers had increased daily urinary potassium excretion rate in relation to their renin levels compared with controls suggesting increased ENaC activity, although in vitro they did not show increased channel activity. Of the common polymorphisms of the RAS studied, angiotensin II receptor type I (AGTR1) 1166 A/C polymorphism was associated with modest changes in RAS activity. Thus, patients homozygous for the C allele tended to have increased aldosterone and decreased renin levels. In vitro functional studies using transfected HEK293 cells provided additional evidence that the AGTR1 1166 C allele may be associated with increased expression of the AGTR1. Common polymorphisms of the alpha-adducin and the RAS genes did not significantly predict BP responses to one-month monotherapies with hydroclorothiazide, bisoprolol, amlodipin, or losartan. In conclusion, two novel mutations of ENaC subunits causing Liddle’s syndrome were identified. In addition, three common ENaC polymorphisms were shown to be associated with occurrence of essential hypertension, but their exact functional and clinical consequences remain to be explored. The AGTR1 1166 C allele may modify the endocrine phenotype of hypertensive patients, when present in homozygous form. Certain widely studied polymorphisms of the ACE, angiotensinogen, AGTR1 and alpha-adducin genes did not significantly affect responses to a thiazide, β-blocker, calcium channel antagonist, and angiotensin II receptor antagonist.

Prospecting for alternate sources of shikimic acid, a precursor of Tamiflu, a bird-flu drug

Shikimic acid, more commonly known by its anionic form, shikimate, is an important intermediate compound of the ‘shikimate pathway’ in plants and microorganisms1. It is the principal precursor for the synthesis of aromatic amino acids, phenylalanine, tryptophan and tyrosine and other compounds such as alkaloids, phenolics and phenyl propanoids2. It is used extensively as a chiral building block for the synthesis of a number of compounds in both pharmaceutical and cosmetic industries3. In the recent past, the focus on shikimic acid has increased since it is the key precursor for the synthesis of Tamiflu, the only drug against avian flu caused by the H5N1 virus4,5. Shikimic acid is converted to a diethyl ketal intermediate, which is then reduced in two steps to an epoxide that is finally transformed to Tamiflu6.

Effect of the headgroup variation on the gene transfer properties of cholesterol based cationic lipids possessing ether linkage

Eight cholesterol based cationic lipids differing in the headgroup have been synthesized based on the ether linkage between the cationic headgroup and the cholesterol backbone. All the lipids formed stable suspensions in water. Transfection efficacies were examined in the absence and presence of serum using their optimized liposomal (lipid:DOPE) formulations. Our results showed that the transfection activities depend on the nature of the headgroup. Lipid bearing 4-N,N′-dimethylaminopyridine (DMAP) as headgroup showed the maximum transfection efficacy in the presence of serum. Importantly, the optimized formulation for this cationic lipid does not require DOPE, which is being used by most commercially available formulations. Cytotoxicity studies showed that the introduction of the positive charge decreases the cell viability of the cationic lipid formulations. Gel electrophoresis and Ethidium bromide exclusion assay revealed the different DNA binding abilities of formulations depending upon the headgroup of the cholesteryl lipid.

Anti-TNF treatment in juvenile idiopathic arthritis and associated uveitis : Clinical perspectives on growth, uveitis, and drug survival

Juvenile idiopathic arthritis (JIA) is a heterogeneous group of childhood chronic arthritides, associated with chronic uveitis in 20% of cases. For JIA patients responding inadequately to conventional disease-modifying anti-rheumatic drugs (DMARDs), biologic therapies, anti-tumor necrosis factor (anti-TNF) agents are available. In this retrospective multicenter study, 258 JIA-patients refractory to DMARDs and receiving biologic agents during 1999-2007 were included. Prior to initiation of anti-TNFs, growth velocity of 71 patients was delayed in 75% and normal in 25%. Those with delayed growth demonstrated a significant increase in growth velocity after initiation of anti-TNFs. Increase in growth rate was unrelated to pubertal growth spurt. No change was observed in skeletal maturation before and after anti-TNFs. The strongest predictor of change in growth velocity was growth rate prior to anti-TNFs. Change in inflammatory activity remained a significant predictor even after decrease in glucocorticoids was taken into account. In JIA-associated uveitis, impact of two first-line biologic agents, etanercept and infliximab, and second-line or third-line anti-TNF agent, adalimumab, was evaluated. In 108 refractory JIA patients receiving etanercept or infliximab, uveitis occurred in 45 (42%). Uveitis improved in 14 (31%), no change was observed in 14 (31%), and in 17 (38%) uveitis worsened. Uveitis improved more frequently (p=0.047) and frequency of annual uveitis flares was lower (p=0.015) in those on infliximab than in those on etanercept. In 20 patients taking adalimumab, 19 (95%) had previously failed etanercept and/or infliximab. In 7 patients (35%) uveitis improved, in one (5%) worsened, and in 12 (60%) no change occurred. Those with improved uveitis were younger and had shorter disease duration. Serious adverse events (AEs) or side-effects were not observed. Adalimumab was effective also in arthritis. Long-term drug survival (i.e. continuation rate on drug) with etanercept (n=105) vs. infliximab (n=104) was at 24 months 68% vs. 68%, and at 48 months 61% vs. 48% (p=0.194 in log-rank analysis). First-line anti-TNF agent was discontinued either due to inefficacy (etanercept 28% vs. infliximab 20%, p=0.445), AEs (7% vs. 22%, p=0.002), or inactive disease (10% vs. 16%, p=0.068). Females, patients with systemic JIA (sJIA), and those taking infliximab as the first therapy were at higher risk for treatment discontinuation. One-third switched to the second anti-TNF agent, which was discontinued less often than the first. In conclusion, in refractory JIA anti-TNFs induced enhanced growth velocity. Four-year treatment survival was comparable between etanercept and infliximab, and switching from first-line to second-line agent a reasonable therapeutic option. During anti-TNF treatment, one-third with JIA-associated anterior uveitis improved.

Sitagliptin and other ‘gliptins’ - why prescribe them?

Introduction In 2008, the Federal Drug Administration (FDA) required all new glucose-lowering therapies to show cardiovascular safety, and this applies to the dipeptidyl peptidase (DPP)-4 inhibitors (‘gliptins’). At present, there is contradictory evidence on whether the gliptins increase hospitalizations for heart failure. Areas covered This is an evaluation of the Trial Evaluating Cardiovascular Outcomes with Sitagliptin (TECOS) in high risk cardiovascular subjects with type 2 diabetes [1]. TECOS demonstrated non-inferiority for sitagliptin over placebo for the primary outcome, which was cardiovascular death, nonfatal myocardial infarction, nonfatal stroke, or hospitalization for unstable angina. There was no difference in the rate of hospitalization for heart failure between sitagliptin and placebo. Expert Opinion Despite the results of TECOS, debate over the effects of sitagliptin on the rates of hospitalizations for heart failure continues with some recent studies suggesting increased rates. Recently, empagliflozin (an inhibitor of sodium-glucose cotransporter 2) has been shown to reduce cardiovascular outcomes in subjects with type 2 diabetes, including the rates of hospitalization for heart failure. In our opinion, these positive findings with empagliflozin suggest that it should be prescribed in preference to the gliptins, including sitagliptin, unless any positive cardiovascular outcomes are reported for the gliptins.

Long-term effects of fenofibrate on lipoproteins and surrogate markers of macrovascular and microvascular disease in people with type 2 diabetes

Background and aims. Diabetic dyslipidemia is a highly atherogenic triad of increased triglycerides, decreased HDL cholesterol, and small dense LDL. Fibrates have a beneficial effect on diabetic dyslipidemia, and they have reduced cardiovascular events in randomized trials. Fenofibrate has reduced albuminuria and markers of low-grade inflammation and endothelial dysfunction. The present studies were undertaken to characterize the alterations of VLDL and LDL subclasses and to investigate the binding of LDL to arterial wall in type 2 diabetes. Further purpose was to elucidate the effects of fenofibrate on several lipoprotein subclasses, augmentation index (AIx), carotid intima-media thickness (IMT), and renal function. Subjects. 239 type 2 diabetic subjects were recruited among participants of the FIELD (Fenofibrate Intervention and Event Lowering in Diabetes) study at the Helsinki centre. The patients were randomized to fenofibrate (200mg/d) or placebo for 5 years. Additionally, a healthy control group (N = 93) was recruited. Results. VLDL1 triglycerides increased in similar proportion to total triglycerides in type 2 diabetic patients and control subjects. Despite the increase in total apoCIII levels, VLDL apoCIII was decreased in diabetic patients. Enrichment of LDL with apoCIII induced a small increase in binding of LDL to arterial wall proteoglycan. Intrinsic characteristics of diabetic LDL, rather than levels of apoCIII, were responsible for increased proteoglycan binding of diabetic LDL with high apoCIII. Fenofibrate reduced triglycerides, increased LDL size, and shifted HDL subclasses towards smaller particles with no change in levels of HDL cholesterol. High levels of homocysteine were associated with lower increase of HDL cholesterol and apoA-I during fenofibrate treatment. Long-term fenofibrate treatment did not improve IMT, AIx, inflammation, or endothelial function. Fenofibrate decreased creatinine clearance and estimated glomerular filtration rate. No effect on albuminuria was seen with fenofibrate. Instead, Cystatin C was increased during fenofibrate treatment. Conclusions. 1) Elevation of VLDL 1 triglycerides was the major determinant of plasma triglyceride concentration in control subjects and type 2 diabetic patients. 2) LDL with high apoCIII showed multiple atherogenic properties, that were only partially mediated by apoCIII per se in type 2 diabetes 3) Fenofibrate demonstrated no effect on surrogate markers of atherosclerosis. 4) Fenofibrate had no effect on albuminuria and the observed decrease in markers of renal function could complicate the clinical surveillance of the patients. 5) Fenofibrate can be used to treat severe hypertriglyceridemia or in combination therapy with statins, but not to increase HDL levels.

Detection and progression of chronic allograft nephropathy : A study based on protocol biopsies

Background. Kidney transplantation (KTX) is considered to be the best treatment of terminal uremia. Despite improvements in short-term graft survival, a considerable number of kidney allografts are lost due to the premature death of patients with a functional kidney and to chronic allograft nephropathy (CAN). Aim. To investigate the risk factors involved in the progression of CAN and to analyze diagnostic methods for this entity. Materials and methods. Altogether, 153 implant and 364 protocol biopsies obtained between June 1996 and April 2008 were analyzed. The biopsies were classified according to Banff ’97 and chronic allograft damage index (CADI). Immunohistochemistry for TGF-β1 was performed in 49 biopsies. Kidney function was evaluated by creatinine and/or cystatin C measurement and by various estimates of glomerular filtration rate (GFR). Demographic data of the donors and recipients were recorded after 2 years’ follow-up. Results. Most of the 3-month biopsies (73%) were nearly normal. The mean CADI score in the 6-month biopsies decreased significantly after 2001. Diastolic hypertension correlated with ΔCADI. Serum creatinine concentration at hospital discharge and glomerulosclerosis were risk factors for ΔCADI. High total and LDL cholesterol, low HDL and hypertension correlated with chronic histological changes. The mean age of the donors increased from 41 -52 years. Older donors were more often women who had died from an underlying disease. The prevalence of delayed graft function increased over the years, while acute rejections (AR) decreased significantly over the years. Sub-clinical AR was observed in 4% and it did not affect long-term allograft function or CADI. Recipients´ drug treatment was modified along the Studies, being mycophenolate mophetil, tacrolimus, statins and blockers of the renine-angiotensin-system more frequently prescribed after 2001. Patients with a higher ΔCADI had lower GFR during follow-up. CADI over 2 was best predicted by creatinine, although with modest sensitivity and specificity. Neither cystatin C nor other estimates of GFR were superior to creatinine for CADI prediction. Cyclosporine A toxicity was seldom seen. Low cyclosporin A concentration after 2 h correlated with TGF- β1 expression in interstitial inflammatory cells, and this predicted worse graft function. Conclusions. The progression of CAN has been affected by two major factors: the donors’ characteristics and the recipients’ hypertension. The increased prevalence of DGF might be a consequence of the acceptance of older donors who had died from an underlying disease. Implant biopsies proved to be of prognostic value, and they are essential for comparison with subsequent biopsies. The progression of histological damage was associated with hypertension and dyslipidemia. The augmented expression of TGF-β1 in inflammatory cells is unclear, but it may be related to low immunosuppression. Serum creatinine is the most suitable tool for monitoring kidney allograft function on every-day basis. However, protocol biopsies at 6 and 12 months predicted late kidney allograft dysfunction and affected the clinical management of the patients. Protocol biopsies are thus a suitable surrogate to be used in clinical trials and for monitoring kidney allografts.

Ultrasound-Triggered Controlled Drug Delivery and Biosensing Using Silica Nanotubes

Silica nanotubes (SNTs) have been demonstrated here as a versatile host for controlled drug delivery and biosensing. The sol-gel template synthesized SNTs have a slow rate of drug release. Application of an external stimulus in the form of ultrasound to or chemical functionalization of synthesized SNT results in higher yield of drug release as well as yield of drug release varying linearly with time. In case of controlled drug delivery triggered by ultrasound, drug yield as function of time is found to be heavily dependent on the ultrasound impulse protocol. Impulses of shorter duration (similar to 0.5 min) and shorter time intervals between successive impulses resulted in higher drug yields. Confinement of hemoglobin (Hb) inside nanometer sized channels of SNT does not have any detrimental effect on the native protein structure and function. Observance of significant enhancement in direct electron transfer of Hb makes the SNTs also promising for application in biosensors.