Safety and Feasibility of a Novel Dosing Regimen of Tirofiban Administered in Patients with Acute Myocardial Infarction with ST Elevation Before Primary Coronary Angioplasty: A Pilot Study

BACKGROUND: Intravenous glycoprotein GP IIb/IIIa receptor antagonists administered to patients with acute coronary syndromes limit platelet-dependent thrombus formation and vasoconstriction and lower the complication rate of PCI. The efficacy of glycoprotein IIb/IIIa inhibitors critically depends on appropriate suppression of platelet aggregation. A growing body of evidence indicates that regimen of tirofiban used in several recent trials may be suboptimal. We investigated if a novel regimen of dosage of tirofiban administered to patients with acute myocardial infarction with ST elevation (STEMI) before primary angioplasty is safe, feasible and whether such treatment improves coronary flow in infarct-related artery. METHODS: It was an open-label, non-randomized, prospective observational study. 253 consecutive patients with STEMI, qualified to PCI were included. 104 of patients (group 1) received heparin plus tirofiban at a novel regimen (10 microg/kg bolus, followed by 0.4 microg/kg/min for 30 min and then 0.1 microg/kg/min for 12-24 hours) and the remaining 149 of the patients (group 2) received a standard dose of heparin prior to PCI. Bleeding complications were recorded. The primary end point of the study was combined TIMI 1 + 2 + 3 grade flow at the time of first contrast medium injection during angiography for primary PCI. RESULTS: Heparin was administered 50.3 +/- 58.1 minutes (group 1) or 62.3 +/- 67.3 minutes (group 2) ( p = 0.205). Tirofiban was administered for an average of 14.5 +/- 14.4 minutes before TIMI assessment (group 1). In patients treated with heparin + tirofiban the rate of combined TIMI 1 + 2 + 3 coronary flow was higher (38.4% vs. 24.8%, p = 0.020) as compared to patients treated with heparin alone. The difference in the rate of TIMI > or = 2 coronary blood flow between the groups 1 and 2 (24.0% vs. 20.1%) has not reached statistical significance ( p = 0.459). At the same time the significant difference in the rate of TIMI 1 coronary blood flow between the groups 1 and 2 was noted (14.4 vs. 4.7%, p = 0.007). In hospital mortality in the groups 1 and 2 was similar (5.3 vs. 4.8%, p = 0.838). Significant difference was noted between the groups 1 and 2 with regard to minor bleeding complications (17.3 vs. 8.7%, p = 0.041). CONCLUSION: In patients undergoing primary angioplasty for acute myocardial infarction the novel regimen of tirofiban is well tolerated and feasible, and is associated with improvement in coronary blood flow in the infarct related artery. Larger studies assessing the effects of tirofiban on clinical outcomes of patients with AMI undergoing primary angioplasty seem worthwhile.     

Progressive aggregation despite chaperone associations of a mutant SOD1-YFP in transgenic mice that develop ALS

Recent studies suggest that superoxide dismutase 1 (SOD1)-linked amyotrophic lateral sclerosis results from destabilization and misfolding of mutant forms of this abundant cytosolic enzyme. Here, we have tracked the expression and fate of a misfolding-prone human SOD1, G85R, fused to YFP, in a line of transgenic G85R SOD1-YFP mice. These mice, but not wild-type human SOD1-YFP transgenics, developed lethal paralyzing motor symptoms at 9 months. In situ RNA hybridization of spinal cords revealed predominant expression in motor neurons in spinal cord gray matter in all transgenic animals. Concordantly, G85R SOD-YFP was diffusely fluorescent in motor neurons of animals at 1 and 6 months of age, but at the time of symptoms, punctate aggregates were observed in cell bodies and processes. Biochemical analyses of spinal cord soluble extracts indicated that G85R SOD-YFP behaved as a misfolded monomer at all ages. It became progressively insoluble at 6 and 9 months of age, associated with presence of soluble oligomers observable by gel filtration. Immunoaffinity capture and mass spectrometry revealed association of G85R SOD-YFP, but not WT SOD-YFP, with the cytosolic chaperone Hsc70 at all ages. In addition, 3 Hsp110's, nucleotide exchange factors for Hsp70s, were captured at 6 and 9 months. Despite such chaperone interactions, G85R SOD-YFP formed insoluble inclusions at late times, containing predominantly intermediate filament proteins. We conclude that motor neurons, initially “compensated” to maintain the misfolded protein in a soluble state, become progressively unable to do so.     

Human glioma demonstrates cell line specific results with ATP-based chemiluminescent cellular proliferation assays

Alteration of tumor cell growth kinetics is the goal of nearly all current or proposed therapies for human neoplasms. The adenosine triphosphate (ATP) chemiluminescent assay has been used for some time as a surrogate marker of in vitro cell growth. Here we present data showing that three human glioblastoma cell lines (U87, U251, G55) demonstrate significantly different cell number to luminescence relationships when subjected to this assay. We plated progressively increasing numbers of cells per well; from 1000 to 50,000 were grown in Dulbecco’s modified Eagle’s medium without serum and cultured for 6 hours. Cells were then lysed and subjected to the chemiluminescent assay to measure ATP levels and a linear relationship between cell number and measured luminescence was found. Despite this, we found that the slope of the regression line (β) varied markedly between different cell lines (U251 [β = 0.968 ± 0.3] vs. U87 [β = 0.772 ± 0.2] vs. G55 [β = 0.757 ± 0.2]; p < 0.0001), suggesting a difference in ATP luminescence per cell between these cell lines. Thus, we have demonstrated that luminescence values are internally linear within a given cell population, but luminescence level per cell varies significantly between different glioma cell lines. Our findings suggest that different glioma cell lines have unique levels of ATP per cell.     

Mutations in the Nijmegen breakage syndrome gene in medulloblastomas

PURPOSE: Cerebellar medulloblastoma is a highly malignant, invasive embryonal tumor with preferential manifestation in children. Nijmegen breakage syndrome (NBS) with NBS1 germ-line mutations is a rare autosomal recessive disease with clinical features that include microcephaly, mental and growth retardation, immunodeficiency, increased radiosensitivity, and predisposition to cancer. There may be functional interactions between NBS1 and the TP53 pathways. The objective of the present study is to assess whether NBS1 mutations play a role in the pathogenesis of sporadic medulloblastomas. EXPERIMENTAL DESIGN: Forty-two cases of medulloblastomas were screened for mutations in the NBS1 gene (all 16 exons) and the TP53 gene (exons 5-8) by single-stranded conformational polymorphism followed by direct DNA sequencing. RESULTS: Seven of 42 (17%) medulloblastomas carried a total of 15 NBS1 mutations. Of these, 10 were missense point mutations and 5 were intronic splicing mutations. None of these were reported previously as germ-line mutations in NBS patients. No NBS1 mutations were detected in peritumoral brain tissues available in two patients. Of 5 medulloblastomas with TP53 mutations, 4 (80%) contained NBS1 mutations, and there was a significant association between TP53 mutations and NBS1 mutations (P = 0.001). CONCLUSIONS: We provide evidence of medulloblastomas characterized by NBS1 mutations typically associated with mutational inactivation of the TP53 gene.     

High plasma adenine concentration in chronic renal failure and its relation to erythrocyte ATP

BACKGROUND: Concentration of plasma adenine has been found to increase in chronic renal failure (CRF). The aim of the present study was to evaluate whether high plasma adenine concentration contributes to the elevated ATP in erythrocytes of patients with CRF. METHODS: Three groups of patients with CRF were studied: (A) 30 patients with different degree of CRF; (B) 11 patients on hemodialysis, and (C) 12 patients after successful renal transplantation. Concentrations of plasma adenine and erythrocyte adenine nucleotides were measured in groups A, B and C. Furthermore, adenine incorporation into erythrocyte adenine nucleotide pool was measured in group A. RESULTS: A positive correlation between plasma adenine and creatinine concentrations was found in CRF as well as between plasma adenine and erythrocyte ATP. Furthermore, positive correlation was evident between the rate of adenine incorporation into erythrocyte adenine nucleotide pool and the severity of CRF. A significant reduction in both plasma adenine and erythrocyte ATP was observed immediately following hemodalysis, but 2 days later, high predialysis plasma adenine and erythrocyte ATP concentrations were restored. Following successful renal transplantation erythrocyte ATP and plasma adenine concentrations reached control values. CONCLUSION: Our results provide evidence that plasma adenine concentration increases in parallel to the progress of the disease and that it could be responsible for the increase in erythrocyte ATP of patients with CRF.     

Influence of spontaneous platelet aggregation on progression of glomerular disease

Platelet secretion products may play an important role in the pathogenesis and progression of the kidney disease. Amongst the parameters describing platelet hyperactivity the measurement of spontaneous platelet aggregation (SPA) seems particularly useful. In this study SPA as well as mean platelet volume (MPV), modal platelet volume (PLT Mode) and platelet count (PLT) were investigated in 60 patients with biopsy proven primary glomerulonephritis. SPA was measured using the turbidimetric method according to Born with no enhancers added. Serum creatinine concentration (Cr), reciprocal serum creatinine concentration (1/Cr) and endogenous creatinine clearance (Cl(Cr)) were used for the renal function estimation. Protein and lipid profiles as well as coagulo-fibrinolytic balance were measured in parallel. The investigated group consisted of 30 non-nephrotic patients (CGN) - in 9, SPA was found (CGN-B) while 21 had SPA <10% (CGN-A), and 30 nephrotic patients (CGN+NS) - 19 with SPA (CGN+NS-B) and 11 without (CGN+NS-A). SPA was found to be a constant platelet feature in patients with chronic glomerulopathy. The group remained under observation for 36 months. 41 patients were included in the 3-year prospective study which revealed the significant influence of the blood platelet hyperaggregability on the renal disease progression. A significantly increased serum creatinine concentration, decreased 1/Cr parameter and decreased glomerular filtration rate (Cl(Cr)) were noted in subgroups showing SPA. A significant correlation between SPA and (Delta)Cr/month (r = 0.41), (Delta)1/Cr/month (r = 0.38) as well as (Delta)Cl(Cr)/month (r = 0.52) was found. The platelet activity and thus SPA can be altered by various factors: albumin and fibrinogen plasma concentrations, thrombosis activation and possibly lipoprotein metabolism disturbances. A characteristic feature of spontaneously aggregating platelet is their increased volume (MPV). CONCLUSION: Platelet hyperaggregation in one of nonimmunological factors stimulates the progression of glomerulonephritis.     

Phase I pharmacodynamic study of time and sequence dependency of hydroxyurea in combination with gemcitabine: a California Cancer Consortium Trial

Preclinical studies in our laboratory have demonstrated that prior exposure to hydroxyurea increases the percentage of cells in S phase, enhancing the cytotoxicity of subsequent gemcitabine treatment in human oropharyngeal KB cells. To evaluate the clinical implications of this time- and sequence-dependent potentiation, we performed a phase I trial of hydroxyurea given over 24 h followed by a 30-min infusion of gemcitabine in weeks 1 and 2 of a 3-week cycle. The dose of hydroxyurea was fixed at 500 mg orally every 6 h for four doses starting 24 h before each dose of gemcitabine. The initial dose level of gemcitabine was 250 mg/m(2) on days 2 and 9, and this was escalated stepwise to 1000 mg/m(2) on days 2 and 9. Gemcitabine pharmacokinetics were determined on days 2 and 9 of the first cycle. Of 27 patients enrolled (12 female, 15 male), 24 were evaluable for response and 23 were evaluable for toxicity. Their median age was 56 years (range 27-76 years). Tumor types included lung, head and neck, pancreas, breast, colon, prostate, stomach, ovary, esophagus, germ cell, thyroid, gallbladder, and unknown primary. A total of 80 cycles of treatment were completed. One patient (unknown primary) had an objective partial response lasting 21 months, and 12 patients had stable disease. All observed dose-limiting toxicities were related to myelosuppression. The gemcitabine maximum tolerated dose was established at 750 mg/m(2) on days 2 and 9. Hydroxyurea had no effect on the plasma pharmacokinetics of gemcitabine. These results suggest that hydroxyurea followed by gemcitabine can be safely administered and has activity on this schedule. We are presently developing a phase II trial of this regimen for patients with platinum-resistant head and neck cancer.