Safety, tolerability, and efficacy of fixed combination therapy with dorzolamide hydrochloride 2% and timolol maleate 0.5% in glaucoma and ocular hypertension.

Glaucoma is a collection of diseases characterized by multifactorial progressive changes leading to visual field loss and optic neuropathy most frequently due to elevated intraocular pressure (IOP). The goal of treatment is the lowering of the IOP to prevent additional optic nerve damage. Treatment usually begins with topical pharmacological agents as monotherapy, progresses to combination therapy with agents from up to 4 different classes of IOP-lowering medications, and then proceeds to laser or incisional surgical modalities for refractory cases. The fixed combination therapy with the carbonic anhydrase inhibitor dorzolamide hydrochloride 2% and the beta blocker timolol maleate 0.5% is now available in a generic formulation for the treatment of patients who have not responded sufficiently to monotherapy with beta adrenergic blockers. In pre- and postmarketing clinical studies, the fixed combination dorzolamide-timolol has been shown to be safe and efficacious, and well tolerated by patients. The fixed combination dorzolamide-timolol is convenient for patients, reduces their dosing regimen with the goal of increasing their compliance, reduces the effects of "washout" when instilling multiple drops, and reduces the preservative burden by reducing the number of drops administered per day.

THE HISTONE DEACETYLASE INHIBITOR, MS-275, SENSITIZES METASTATIC OSTEOSARCOMA TO FASL-INDUCED CELL DEATH: A ROLE FOR C-FLIP

The purpose of this study was to determine the effects of the histone deacetylase inhibitor, MS-275, on the Fas signaling pathway and susceptibility of osteosarcoma (OS) to Fas ligand (FasL)-induced cell death. OS metastasizes almost exclusively to the lungs. We have shown that Fas expression in OS cells is inversely correlated with their metastatic potential. Fas+ cells are rapidly eliminated when they enter the lungs via interaction with FasL, which is constitutively expressed in the lungs. Fas- OS cells escape this FasL-induced apoptosis and survive in the lung microenvironment. Moreover, upregulation of Fas in established OS lung metastases results in tumor regression. Therefore, agents that upregulate Fas expression or activate the Fas signaling pathway may have therapeutic potential. Treatment of Fas- metastatic OS cell lines with 2 μM MS-275 sensitized cells to FasL-induced cell death in vitro. We found that MS-275 did not alter the expression of Fas on the cell surface; rather it resulted in increased levels of Fas within the membrane lipid rafts, as demonstrated by an increase in Fas expression in detergent insoluble lipid raft fractions. We further demonstrated that following MS-275 treatment, Fas colocalized with GM1+ lipid rafts and that there was a decrease in c-FLIP (cellular FLICE-inhibitory protein) mRNA and protein. Downregulation of c-FLIP correlated with caspase activation and apoptosis induction. Transfection of cells with shRNA to c-FLIP also resulted in the localization of Fas to lipid rafts. These studies indicate that MS-275 sensitizes OS cells to FasL by upregulating the expression of Fas in membrane lipid rafts, which correlated with the downregulation of c-FLIP. Treatment of nu/nu-mice with established OS lung metastases with oral MS-275 resulted in increased apoptosis, a significant inhibition of c-FLIP expression in tumors and tumor regression. Histopathological examination of mice showed no significant organ toxicity. Overall, these results suggest that the mechanism by which MS-275 sensitizes OS cells and lung metastases to FasL-induced cell death may be by a reduction in the expression of c-FLIP.

Plasminogen activator inhibitor-1, monocyte chemoattractant protein-1, e-selectin and C-reactive protein levels in response to 4-week very-high-fructose or -glucose diets.

BACKGROUND/OBJECTIVES High intake of added sweeteners is considered to have a causal role in the pathogenesis of cardiometabolic disorders. Especially, high-fructose intake is regarded as potentially harmful to cardiometabolic health. It may cause not only weight gain but also low-grade inflammation, which represents an independent risk factor for developing type 2 diabetes and cardiovascular disease. In particular, fructose has been suggested to induce plasminogen activator inhibitor-1 (PAI-1) expression in the liver and to increase circulating inflammatory cytokines. We therefore aimed to investigate, whether high-fructose diet has an impact on PAI-1, monocyte chemoattractant protein-1 (MCP-1), e-selectin and C-reactive protein (CRP) concentrations in healthy humans. SUBJECTS/METHODS We studied 20 participants (12 males and 8 females) of the TUebingen FRuctose Or Glucose study. This is an exploratory, parallel, prospective, randomized, single-blinded, outpatient, hypercaloric, intervention study. The participants had a mean age of 30.9 ± 2.1 years and a mean body mass index of 26.0 ± 0.5 kg/m(2) and they received 150 g of either fructose or glucose per day for 4 weeks.Results:There were neither significant changes of PAI-1, MCP-1, e-selectin and CRP after fructose (n=10) and glucose (n=10) intervention nor treatment effects (all P>0.2). Moreover, we did not observe longitudinal associations of the inflammatory parameters with triglycerides, liver fat, visceral fat and body weight in the fructose group. CONCLUSIONS Temporary high-fructose intake does not seem to cause inflammation in apparently healthy people in this secondary analysis of a small feeding trial.

Furosemide inhibits 11beta-hydroxysteroid dehydrogenase type 2.

11Beta-hydroxsteroid dehydrogenase 2 (11beta-OHSD2) protects the nonselective renal mineralocorticoid receptor from the endogenous glucocorticoid cortisol. Thus, drugs inhibiting 11beta-OHSD2 might enhance urinary loss of potassium. As diuretics influence the renal handling of potassium, we analyzed the impact of 13 commonly used diuretics on 11beta-OHSD2. Furosemide was the only inhibitor. Its inhibition constant (Ki) was 30 micromol when extracts from COS-1 cells transfected with human 11beta-OHSD2 were used as an enzyme source. The type of inhibition was competitive. To establish whether furosemide inhibits 11beta-OHSD2 and 11beta-OHSD1 in the renal target tissue, isolated tubular segments from rats were analyzed. Furosemide decreased the oxidative activity of 11beta-OHSD2 in intact distal tubules and 11beta-OHSD1 in proximal convoluted tubules. For the assessment of furosemide on the excretion of corticosterone metabolites in vivo, rats were given furosemide i.p., and the ratio of tetrahydrocorticosterone plus 5alpha-tetrahydrocorticosterone to 11-dehydrotetrahydrocorticosterone was determined in urine. This ratio increased after the administration of furosemide in all animals, indicating inhibition of the oxidative activity of 11beta-OHSD. Thus, furosemide inhibits the 11beta-OHSD2 enzyme in the target tissue and might by that mechanism enhance the mineralocorticoid effect of 11beta-hydroxyglucocorticoids.

In pneumococcal meningitis a novel water-soluble inhibitor of matrix metalloproteinases and TNF-alpha converting enzyme attenuates seizures and injury of the cerebral cortex

Matrix metalloproteinases (MMPs) and TNF-alpha converting enzyme (TACE) contribute to the pathophysiology of bacterial meningitis. To date, MMP-inhibitors studied in models of meningitis were compromised by their hydrophobic nature. We investigated the pharmacokinetics and the effect of TNF484, a water-soluble hydroxamate-based inhibitor of MMP and TACE, on disease parameters and brain damage in a neonatal rat model of pneumococcal meningitis. At 1 mg/kg q6h TNF484 reduced soluble TNF-alpha and the collagen degradation product hydroxyproline in the cerebrospinal fluid. Clinically, TNF484 attenuated the incidence of seizures and was neuroprotective in the cortex. Water-soluble MMP-inhibitors may hold promise in the therapy of bacterial meningitis.

Alphaviruses induce apoptosis in Bcl-2-overexpressing cells: evidence for a caspase-mediated, proteolytic inactivation of Bcl-2

Bcl-2 oncogene expression plays a role in the establishment of persistent viral infection by blocking virus-induced apoptosis. This might be achieved by preventing virus-induced activation of caspase-3, an IL-1beta-converting enzyme (ICE)-like cysteine protease that has been implicated in the death effector phase of apoptosis. Contrary to this model, we show that three cell types highly overexpressing functional Bcl-2 displayed caspase-3 activation and underwent apoptosis in response to infection with alphaviruses Semliki Forest and Sindbis as efficiently as vector control counterparts. In all three cell types, overexpressed 26 kDa Bcl-2 was cleaved into a 23 kDa protein. Antibody epitope mapping revealed that cleavage occurred at one or two target sites for caspases within the amino acid region YEWD31 (downward arrow) AGD34 (downward arrow) A, removing the N-terminal BH4 region known to be essential for the death-protective activity of Bcl-2. Preincubation of cells with the caspase inhibitor Z-VAD prevented Bcl-2 cleavage and partially restored the protective activity of Bcl-2 against virus-induced apoptosis. Moreover, a murine Bcl-2 mutant having Asp31, Asp34 and Asp36 substituted by Glu was resistant to proteolytic cleavage and abrogated apoptosis following virus infection. These findings indicate that alphaviruses can trigger a caspase-mediated inactivation of Bcl-2 in order to evade the death protection imposed by this survival factor.

Everolimus is a potent inhibitor of activated hepatic stellate cell functions in vitro and in vivo , while demonstrating anti-angiogenic activities

Progression of liver fibrosis to HCC (hepatocellular carcinoma) is a very complex process which involves several pathological phenomena, including hepatic stellate cell activation, inflammation, fibrosis and angiogenesis. Therefore inhibiting multiple pathological processes using a single drug can be an effective choice to curb the progression of HCC. In the present study, we used the mTOR inhibitor everolimus to observe its effect on the in vitro activation of hepatic stellate cells and angiogenesis. The results of the present study demonstrated that everolimus treatment blocked the functions of the immortalized human activated hepatic stellate cell line LX-2 without affecting the viability and migration of primary human stellate cells. We also observed that treatment with everolimus (20 nM) inhibited collagen production by activated stellate cells, as well as cell contraction. Everolimus treatment was also able to attenuate the activation of primary stellate cells to their activated form. Angiogenesis studies showed that everolimus blocked angiogenesis in a rat aortic ring assay and inhibited the tube formation and migration of liver sinusoidal endothelial cells. Finally, everolimus treatment reduced the load of tumoral myofibroblasts in a rat model of HCC. These data suggest that everolimus targets multiple mechanisms, making it a potent blocker of the progression of HCC from liver fibrosis.

Localized Epidermal Cysts as a Radiation Recall Phenomenon in a Melanoma Patient Treated with Radiotherapy and the BRAF Inhibitor Vemurafenib

BRAF inhibitors are broadly used for metastatic melanoma with BRAF mutations. Their use results in various cutaneous side effects, such as the development of keratoacanthomas and squamous cell carcinomas. We report a patient with metastatic melanoma treated with vemurafenib who developed dozens of histologically confirmed epidermal cysts within 2 months after initiation of vemurafenib administration. The cystic lesions were observed only in the localized area where a large exophytic melanoma tumor mass had been previously irradiated. Localized epidermal cysts may constitute an unusual radiation recall reaction in patients treated with BRAF inhibitors.

Relationship between cobalamin-dependent metabolites and both serum albumin and alpha1 -proteinase inhibitor concentrations in hypocobalaminemic dogs of 7 different breeds.

BACKGROUND Increased serum concentrations of homocysteine (HCY) and methylmalonic acid (MMA), the 2 main cobalamin-dependent metabolites, as well as decreased serum albumin and canine alpha1 -proteinase inhibitor (cα1 -PI) concentrations have previously been described in hypocobalaminemic dogs with gastrointestinal disease. However, no studies have been conducted to evaluate potential relationships between these serum biomarkers. OBJECTIVE The aim of this study was to evaluate the relationship between HCY and MMA, 2 cobalamin-dependent metabolites, and both serum albumin and cα1 -PI concentrations in hypocobalaminemic dogs. METHODS Serum samples from 285 dogs including 7 different breeds (Beagle, Boxer, Cocker Spaniel, German Shepherd, Labrador Retriever, Chinese Shar-Pei, and Yorkshire Terrier) with hypocobalaminemia were used. Serum HCY, MMA, albumin, and cα1 -PI concentrations were determined. RESULTS There was a significant correlation between serum HCY and albumin concentrations, as well as serum HCY and cα1 -PI concentrations (ρ = 0.62 and ρ = 0.37, respectively; P < .0001). No correlations were observed between serum MMA and albumin concentrations, or cα1 -PI concentrations (ρ = 0.01 and ρ = 0.08, respectively; P > .05). In addition, significant breed-specific correlations were observed between serum MMA and albumin concentrations in German Shepherds, and serum HCY and MMA concentrations in Chinese Shar-Peis with hypocobalaminemia. CONCLUSIONS This study shows a correlation between serum albumin and cα1 -PI and HCY concentrations, but not with serum MMA concentration in dogs with hypocobalaminemia. In addition, significant breed-specific correlations were observed between serum MMA and albumin concentrations in German Shepherds, as well as serum HCY and MMA concentrations in Chinese Shar-Peis, emphasizing the unique metabolic interactions in those dog breeds affected by hypocobalaminemia.

Bcl-2 predicts response to neoadjuvant chemotherapy and is overexpressed in lymph node metastases of urothelial cancer of the bladder

PURPOSE To assess whether Bcl-2, an inhibitor of the apoptotic cascade, can predict response to neoadjuvant chemotherapy in patients with urothelial cancer of the bladder (UCB). METHODS Bcl-2 expression was analyzed in 2 different tissue microarrays (TMAs). One TMA was constructed of primary tumors and their corresponding lymph node (LN) metastases from 152 patients with chemotherapy-naive UCB treated by cystectomy and pelvic lymphadenectomy (chemotherapy-naive TMA cohort). The other TMA was constructed of tumor samples obtained from 55 patients with UCB before neoadjuvant chemotherapy (transurethral resection of the bladder cancer) and after cystectomy with pelvic lymphadenectomy (residual primary tumor [ypT+], n = 38); residual LN metastases [ypN+], n = 24) (prechemotherapy/postchemotherapy TMA cohort). Bcl-2 overexpression was defined as 10% or more cancer cells showing cytoplasmic immunoreactivity. RESULTS In both TMA cohorts, Bcl-2 overexpression was significantly (P<0.05) more frequent in LN metastases than in primary tumors (chemotherapy-naive TMA group: 18/148 [12%] in primary tumors vs. 39/143 [27%] in metastases; postchemotherapy TMA: ypT+7/35 [20%] vs. ypN+11/19 [58%]). In the neoadjuvant setting, patients with Bcl-2 overexpression in transurethral resection of the bladder cancer specimens showed significantly (P = 0.04) higher ypT stages and less regression in their cystectomy specimens than did the control group, and only one-eighth (13%) had complete tumor regression (ypT0 ypN0). In survival analyses, only histopathological parameters added significant prognostic information. CONCLUSIONS Bcl-2 overexpression in chemotherapy-naive primary bladder cancer is related to poor chemotherapy response and might help to select likely nonresponders.

A novel blood-sparing agent in cardiac surgery? First in-patient experience with the synthetic serine protease inhibitor MDCO-2010: a phase II, randomized, double-blind, placebo-controlled study in patients undergoing coronary artery bypass grafting with cardiopulmonary bypass

BACKGROUND Antifibrinolytics have been used for 2 decades to reduce bleeding in cardiac surgery. MDCO-2010 is a novel, synthetic, serine protease inhibitor. We describe the first experience with this drug in patients. METHODS In this phase II, double-blind, placebo-controlled study, 32 patients undergoing isolated primary coronary artery bypass grafting with cardiopulmonary bypass were randomly assigned to 1 of 5 increasing dosage groups of MDCO-2010. The primary aim was to evaluate pharmacokinetics (PK) with assessment of plasmatic concentrations of the drug, short-term safety, and tolerance of MDCO-2010. Secondary end points were influence on coagulation, chest tube drainage, and transfusion requirements. RESULTS PK analysis showed linear dosage-proportional correlation between MDCO-2010 infusion rate and PK parameters. Blood loss was significantly reduced in the 3 highest dosage groups compared with control (P = 0.002, 0.004 and 0.011, respectively). The incidence of allogeneic blood product transfusions was lower with MDCO-2010 4/24 (17%) vs 4/8 (50%) in the control group. MDCO-2010 exhibited dosage-dependent antifibrinolytic effects through suppression of D-dimer generation and inhibition of tissue plasminogen activator-induced lysis in ROTEM analysis as well as anticoagulant effects demonstrated by prolongation of activated clotting time and activated partial thromboplastin time. No systematic differences in markers of end organ function were observed among treatment groups. Three patients in the MDCO-2010 groups experienced serious adverse events. One patient experienced intraoperative thrombosis of venous grafts considered possibly related to the study drug. No reexploration for mediastinal bleeding was required, and there were no deaths. CONCLUSIONS This first-in-patient study demonstrated dosage-proportional PK for MDCO-2010 and reduction of chest tube drainage and transfusions in patients undergoing primary coronary artery bypass grafting. Antifibrinolytic and anticoagulant effects were demonstrated using various markers of coagulation. MDCO-2010 was well tolerated and showed an acceptable initial safety profile. Larger multi-institutional studies are warranted to further investigate the safety and efficacy of this compound.

Refinement of tourniquet-induced peripheral ischemia/reperfusion injury in rats: comparison of 2 h vs 24 h reperfusion.

Prolonged ischemia of skeletal muscle tissue, followed by reperfusion, leads to ischemia/reperfusion injury (IRI), which is a feared local and systemic inflammatory reaction. With respect to the 3Rs, we wanted to determine which parameters for assessment of IRI require a reperfusion time of 24 h and for which 2 h of reperfusion are sufficient. Rats were subjected to 3 h of hind limb ischemia and 2 h or 24 h of reperfusion. Human plasma derived C1 inhibitor was used as a drug to prevent reperfusion injury. For 2 h of reperfusion the rats stayed under anesthesia throughout (severity grade 1), whereas for 24 h they were awake under analgesia during reperfusion (grade 2). The femoral artery was clamped and a tourniquet was placed, under maintenance of venous return. C1 esterase inhibitor was systemically administered 5 min before the induction of ischemia. No differences in local muscle edema formation and depositions of immunoglobulin G and immunoglobulin M were observed between 2 h and 24 h (P > 0.05), whereas lung edema was only observed after 24 h. Muscle viability was significantly lower after 24 h vs 2 h reperfusion (P < 0.05). Increased plasma creatine kinase (CK)-MM and platelet-derived growth factor (PDGF)-bb could be detected after 2 h, but not after 24 h of reperfusion. By contrast, depositions of C3b/c and fibrin in muscle were only detected after 24 h (P < 0.001). In conclusion, for a first screening of drug candidates to reduce IRI, 2 h reperfusions are sufficient, and these reduce the severity of the animal experiment. Twenty-four-hour reperfusions are only needed for in-depth analysis of the mechanisms of IRI, including lung damage.

Impact of proton pump inhibitor use on magnesium homoeostasis: a cross-sectional study in a tertiary emergency department.

BACKGROUND To date, the use of proton pump inhibitors (PPIs) has been associated with a low risk of hypomagnesaemia and associated adverse outcomes. We hypothesised that a better risk estimate could be derived from a large cohort of outpatients admitted to a tertiary emergency department (ED). METHODS A cross-sectional study was performed in 5118 patients who had measurements of serum magnesium taken on admission to a large tertiary care ED between January 2009 and December 2010. Hypomagnesaemia was defined as a serum magnesium concentration < 0.75 mmol/l. Demographical data, serum electrolyte values, data on medication, comorbidities and outcome with regard to length of hospital stay and mortality were analysed. RESULTS Serum magnesium was normally distributed where upon 1246 patients (24%) were hypomagnesaemic. These patients had a higher prevalence of out-of-hospital PPI use and diuretic use when compared with patients with magnesium levels > 0.75 mmol/l (both p < 0.0001). In multivariable regression analyses adjusted for PPIs, diuretics, renal function and the Charlson comorbidity index score, the association between use of PPIs and risk for hypomagnesaemia remained significant (OR = 2.1; 95% CI: 1.54-2.85). While mortality was not directly related to low magnesium levels (p = 0.67), the length of hospitalisation was prolonged in these patients even after adjustment for underlying comorbid conditions (p < 0.0001). CONCLUSION Use of PPIs predisposes patients to hypomagnesaemia and such to prolonged hospitalisation irrespective of the underlying morbidity, posing a critical concern.

Biochanin A and prunetin improve epithelial barrier function in intestinal CaCo-2 cells via downregulation of ERK, NF-κB, and tyrosine phosphorylation

The single-layered gut epithelium represents the primary line of defense against environmental stressors; thereby monolayer integrity and tightness are essentially required to maintain gut health and function. To date only a few plant-derived phytochemicals have been described as affecting intestinal barrier function. We investigated the impact of 28 secondary plant compounds on the barrier function of intestinal epithelial CaCo-2/TC-7 cells via transepithelial electrical resistance (TEER) measurements. Apart from genistein, the compounds that had the biggest effect in the TEER measurements were biochanin A and prunetin. These isoflavones improved barrier tightness by 36 and 60%, respectively, compared to the untreated control. Furthermore, both isoflavones significantly attenuated TNFα-dependent barrier disruption, thereby maintaining a high barrier resistance comparable to nonstressed cells. In docking analyses exploring the putative interaction with the tyrosine kinase EGFR, these novel modulators of barrier tightness showed very similar values compared to the known tyrosine kinase inhibitor genistein. Both biochanin A and prunetin were also identified as potent reducers of NF-κB and ERK activation, zonula occludens 1 tyrosine phosphorylation, and metalloproteinase-mediated shedding activity, which may account for the barrier-improving ability of these isoflavones.