Differential scanning calorimetry differences in micronized and unmicronized nimesulide uptake processes in biomembrane models.

Nimesulide release from micronized and unmicronized drug particles was tested at pH 7.4 by measuring the transfer to dimyristoylphosphatidylcholine liposomes (multilamellar and unilamellar vesicles), chosen as a biomembrane model. The perturbing effect of increasing molar fractions of pure nimesulide on the thermotropic behaviour of dimyristoylphosphatidylcholine liposomes was investigated by differential scanning calorimetry. In order to study the drug dissolution process by its uptake into void liposomes, measurements were carried out on suspensions of blank liposomes added to weighed amounts of free powdered nimesulide (micronized and unmicronized). The amount of drug transferred was quantified by comparing the effect caused by the dissolved and released drug to that caused by the free drug that had been previously molecularly dissolved in the liposomes. The calorimetric results show that the dissolution rate depends on the nimesulide form (micronized or unmicronized), and that the transfer to the void liposomes is quicker when the drug is in a micronized form. The uptake was faster when unilamellar vesicles were used instead of multilamellar vesicles because of the greater lipid surface. The calorimetric technique could represent an alternative 'in vitro' method that can be applied to the study of the dissolution kinetics directly at the site of drug uptake, mimicking a biological system.     

Prognostic value of CD40 in adult soft tissue sarcomas.

PURPOSE: The purpose is to evaluate the expression of CD40, a membrane protein predominantly expressed on B cells, dendritic cells, and macrophages, in a series of adult soft tissue sarcomas and to test its possible prognostic value. EXPERIMENTAL DESIGN: CD40 expression was studied by immunohistochemistry. Correlations with other baseline characteristics of patients and tumors were analyzed with chi(2) test. The prognostic value was studied with univariable and multivariable analysis adjusted by age, sex, tumor size, grade, location, and distant metastases. RESULTS: Eighty-two patients, between January 1994 and May 2001, were analyzed. Membrane or cytoplasmic staining for CD40 protein was absent in 30% of the tumors but present in <10% of cells in 22 (27%), in 10% to 50% in 23 (28%), and in >50% of cells in 12 (15%) tumors. There was no correlation between CD40 expression and age, sex, size, grade, and location of the primary tumor and distant metastases. With 61 patients (74.4%) progressed and 31 (37.8%) dead, CD40 expression was a significant prognostic factor for disease-free and overall survival at univariable and multivariable analysis. Patients with tumors expressing CD40 in >50% of cells had a dramatically unfavorable prognosis with median disease-free and overall survival of 7 and 17 months, respectively, and hazard ratios of relapse and death as compared with patients with CD40-negative tumors of 2.89 (95% confidence interval: 1.26-6.60) and 6.92 (95% confidence interval: 2.18-22.0), respectively. CONCLUSIONS: These data suggest that expression of CD40 protein in >50% of cells might indicate an unfavorable prognosis in adult soft tissue sarcomas.     

Stroma cells: a novel target of herceptin activity.

PURPOSE: Stroma cells play a relevant role in tumor development and progression. We investigated the activity of herceptin (HER), a humanized monoclonal antibody widely used for the treatment of HER2-overexpressing epithelial cancer, toward stroma cell lines L87/4 and L88/5. EXPERIMENTAL DESIGN: We studied the antiproliferative potential of HER and role of human serum in HER activity. We also investigated the ability of HER to alter ancillary functions of L87/4 and L88/5, such as support to long-term hematopoiesis, growth factor production, breast cancer cell adhesion, and proliferation. RESULTS: Flow cytometry showed that HER2 membrane expression in L87/4 and L88/5 stroma cells was intermediate between the expression in HER2-negative/dim MCF-7 breast cancer cells and HER2-bright SK-BC3 breast cancer cells. HER2 gene amplification was not detected by fluorescence in situ hybridization in either stromal cell lines. HER significantly inhibited L87/4 and L88/5 proliferation. Mean ID(50)s were found to be 2000 and 1700 micro g/ml for L87/4 and L88/5, respectively, after 3-day exposure and 800 micro g/ml for both cell lines after 9-day exposure. The presence of 10% human serum in the culture increased HER inhibitory activity. IC(50) of stroma cells was found to be intermediate between HER2-bright breast cancer cells (SK-BC3) and HER2-negative/dim breast cancer cells (MCF-7). The drug did not significantly affect the ability of stroma cells to support long-term hematopoiesis in the cobblestone area forming cell assay. In contrast, in coculture assay, MCF7 cells demonstrated a worse adhesion and growth capability on HER-treated stroma layers when compared with untreated stroma. Moreover, HER significantly reduced vascular endothelial growth factor production by L88/5 cells. CONCLUSIONS: Our data support the novel finding that HER may have a relevant activity against stroma cells.     

Targeted liposomal c-myc antisense oligodeoxynucleotides induce apoptosis and inhibit tumor growth and metastases in human melanoma models.

PURPOSE: Melanoma is a highly malignant and increasingly common tumor. Because the cure rate of metastatic melanoma by conventional treatment is very low, new therapeutic approaches are needed. We previously reported that coated cationic liposomes (CCL) targeted with a monoclonal antibody against the disialoganglioside (GD(2)) and containing c-myb antisense oligodeoxynucleotides (asODNs) resulted in a selective inhibition of the proliferation of GD(2)-positive neuroblastoma cells in vitro. EXPERIMENTAL DESIGN: Here, we tested the in vivo antitumor effects of this novel antisense liposomal formulation by targeting the c-myc oncogene on melanoma, a neuroectodermal tumor sharing with neuroblastoma the expression of GD(2). RESULTS: Our methods produced GD(2)-targeted liposomes that stably entrapped 90% of added c-myc asODNs. These liposomes showed a selective binding for GD(2)-positive melanoma cells in vitro. Melanoma cell proliferation was inhibited to a greater extent by GD(2)-targeted liposomes containing c-myc asODNs (aGD(2)-CCL-myc-as) than by nontargeted liposomes or free asODNs. The pharmacokinetic results obtained after i.v. injection of [(3)H]-myc-asODNs, free or encapsulated in nontargeted CCLs or GD(2)-targeted CCLs, showed that free c-myc-asODNs were rapidly cleared, with less than 10% of the injected dose remaining in blood at 30 min after injection. c-myc-asODNs encapsulated within either CCL or aGD(2)-CCL demonstrated a more favorable profile in blood, with about 20% of the injected dose of each preparation remaining in vivo at 24 h after injection. In an in vivo melanoma experimental metastatic model, aGD(2)-CCL-myc-as, at a total dose of only 10 mg of asODN per kilogram, significantly inhibited the development of microscopic metastases in the lung compared with animals treated with myc-asODNs, free or entrapped in nontargeted liposomes, or aGD(2)-CCL encapsulating scrambled asODNs (P < 0.01). Moreover, mice bearing established s.c. human melanoma xenografts treated with aGD(2)-CCL-myc-as exhibited significantly reduced tumor growth and increased survival (P < 0.01 versus control mice). The mechanism for the antitumor effects appears to be down-regulation of the expression of the c-myc protein and interruption of c-myc-mediated signaling: induction of p53 and inhibition of Bcl-2 proteins, leading to extensive tumor cell apoptosis. CONCLUSION: These results suggest that inhibition of c-myc proto-oncogene by GD(2)-targeted antisense therapy could provide an effective approach for the treatment of melanoma in an adjuvant setting.     

An Analysis of Approaches to the Management of Endometrial Cancer in North America: A CTF Study

Objective.The aim of this study was to define the clinical–therapeutical approach to endometrial cancer now being followed in some of the most important centers of reference for gynecological cancer in North America by means of a questionnaire.Study design.The questionnaire focused on four principal areas: (1) surgical staging and therapy; (2) adjuvant treatment; (3) treatment modifications; and (4) management of advanced stages (FIGO III–IV).Results.There were 48 evaluable responses (77%) received by the end of December 1994 which were considered for this analysis. Lymphadenectomy is utilized routinely in 26/48 centers (54.2%) and in selective clinical–pathological conditions in another 21/48 centers (43.5%). In the majority of centers (31/48; 64.6%) radical surgery is utilized for selected indications such as cervical involvement. Only 3/48 (6.2%) centers consider the vaginal approach totally inappropriate. The great majority (40/48; 83.3%) of the centers considered postsurgical adjuvant therapy to be necessary in FIGO Stage Ic. Brachytherapy is routinely performed in 3 centers (6.2%) in postsurgical management of Stage I endometrial cancer, while the majority of the centers (31/48; 64.6%) perform brachytherapy of the vaginal vault in certain clinical–pathological conditions. A wide variety of treatments are used for advanced stages (FIGO III–IV).Conclusions.It emerges that some controversial aspects exist on endometrial cancer treatment, and these conflicting data need a large-scale multicenter randomized clinical trial.     

Advanced colorectal cancer in the elderly: results of consecutive trials with 5-fluorouracil-based chemotherapy

To evaluate toxicity and efficacy of chemotherapy in elderly patients (≥65 years of age) with advanced colorectal cancer, data from two consecutive trials conducted between 1984 and 1995 at the National Institute for Cancer Research were analysed comparing the results of treatment in those 65 years of age or older and in those younger than 65 years. Of 215 patients recruited, 82 elderly patients (median age 70 years, median performance status 1) received one of the following regimens based on 5-fluorouracil (5-FU): (1) weekly 5-FU 600 mg/m² i.v. bolus (30 patients); (2) weekly 5-FU 600 mg/m² bolus plus leucovorin (LV) 500 mg/m² 2-h i.v. infusion (28 patients); (3) Weekly 5-FU 2600 mg/m² 24-h continuous i.v. infusion plus LV 100 mg 4-h i.v. infusion and 50 mg orally every 4 h for five doses (24 patients). Overall, 1071 chemotherapy cycles were administered with a median number of 12 courses per patient. The main side effects were diarrhoea, observed in 38% of patients, stomatitis in 24% of patients and hand-foot syndrome in 13% of patients, and haematological toxicity affected only 15% of patients. No patient suffered grade IV toxicity. In three patients chemotherapy was discontinued because of toxicity (two patients suffered grade III diarrhoea, one patient grade III hand-foot syndrome). No significant difference in toxicity was evident between patients older than or younger than 65 years. Analysis of median dose intensity demonstrated no difference between the two groups. Overall objective response was observed in 18% (95% confidence limits 11–29) of elderly patients (15/82) in comparison with 23% (95% CL 17–32) of patients <65 years of age (31/133 pts). In conclusion, chemotherapy in elderly patients with advanced colorectal cancer is a safe and effective treatment with acceptable toxicity and comparable objective response rates. Received: 6 January 1998 / Accepted: 27 February 1998     

From target identification to drug screening assays for neurodegenerative diseases.

Treatment of neurodegenerative diseases represents a major challenge for the pharmaceutical industry. Key to developing novel and efficacious therapeutics is the discovery of new druggable targets. Toward this aim, the current drug discovery process is strongly relying on the improved understanding of disease mechanisms and on a synergistic approach with chemistry, molecular biology and robotics. In this scenario, we present the case of a newly discovered molecular mechanism that may be of interest for drug discovery programmes in Huntington's disease and other neurodegenerative diseases.     

Sequential endo-laparoscopic treatment in patients with common bile calculi

BACKGROUND: Laparoscopic treatment of common bile duct (CBD) stones is gaining great acceptance worldwide, but actually it requires skills and technologies too expensive for a great part of general surgeons. So endoscopic removal of CBD stones before cholecystectomy is usually performed. Since 1991 in our department we started a policy of selective preoperative cholangiopancreatography (ERCP) in patients suspected for choledocholithiasis and waiting for laparoscopic cholecystectomy. METHODS: A retrospective study has been made on a population of 1100 patients who underwent elective laparoscopic cholecystectomy in the period between January 1991 and December 1997. They were 391 male and 719 female with a mean age of 52 years, 126 of whom (11.5%) were selected to have ERCP preoperatively because they had clinical, biochemical and ultrasound signs of the presence of common bile duct stones (CBDS). RESULTS: Successful cannulation of the CBD was achieved in 124 cases (98.4%), with failures due to ampullary diverticula. In 7 cases (5.5%) a precut was necessary to obtain cannulation. Sphincterotomy was performed in 113 patients (89.7%). In 93 patients (73.8%) stones were found (87 macrolithiasis and 6 microlithiasis); in 91 (97.8%) stones were removed in one (87) or two (4) endoscopic session. There were 2 major complications (one bleeding and one severe pancreatitis) due to ERCP or a sphincterotomy. Two patients developed symptoms from unsuspected common bile duct stones after LC and were removed endoscopically. No complications during LC were due to ERCP or ES. CONCLUSIONS: Selective preoperative ERCP is an effective way of clearing the CBD stones before laparoscopic cholecystectomy, with low rate of complications related to endoscopic and laparoscopic procedures, and short mean hospital stay (5.5 days), according to the concept of minimally invasive treatment.     

Impaired Vagal Activity in Long-COVID-19 Patients

Long-COVID-19 refers to the signs and symptoms that continue or develop after the “acute COVID-19” phase. These patients have an increased risk of multiorgan dysfunction, readmission, and mortality. In Long-COVID-19 patients, it is possible to detect a persistent increase in D-Dimer, NT-ProBNP, and autonomic nervous system dysfunction. To verify the dysautonomia hypothesis in Long-COVID-19 patients, we studied heart rate variability using 12-lead 24-h ECG monitoring in 30 Long-COVID-19 patients and 20 No-COVID patients. Power spectral analysis of heart rate variability was lower in Long-COVID-19 patients both for total power (7.46 ± 0.5 vs. 8.08 ± 0.6; p < 0.0001; Cohens-d = 1.12) and for the VLF (6.84 ± 0.8 vs. 7.66 ± 0.6; p < 0.0001; Cohens-d = 1.16) and HF (4.65 ± 0.9 vs. 5.33 ± 0.9; p = 0.015; Cohens-d = 0.76) components. The LF/HF ratio was significantly higher in Long-COVID-19 patients (1.46 ± 0.27 vs. 1.23 ± 0.13; p = 0.001; Cohens-d = 1.09). On multivariable analysis, Long-COVID-19 is significantly correlated with D-dimer (standardized β-coefficient = 0.259), NT-ProBNP (standardized β-coefficient = 0.281), HF component of spectral analysis (standardized β-coefficient = 0.696), and LF/HF ratio (standardized β-coefficient = 0.820). Dysautonomia may explain the persistent symptoms in Long COVID-19 patients. The persistence of a procoagulative state and an elevated myocardial strain could explain vagal impairment in these patients. In Long-COVID-19 patients, impaired vagal activity, persistent increases of NT-ProBNP, and a prothrombotic state require careful monitoring and appropriate intervention.