No effect of acidification or freezing on urinary metanephrine levels.

BACKGROUND: Urinary metanephrine is a reliable method to estimate catecholamine secretion. Traditionally, urinary metanephrines are collected into chilled containers containing hydrochloric acid (HCl) and most laboratories freeze urinary samples before analysis. It is uncertain if these pre-analytic procedures alter metanephrine values. AIM: To evaluate if acidifying and freezing urine samples affect the accuracy of urinary metanephrine measurements. METHODS: Random urine samples from healthy individuals were collected. Urine samples were distributed into two containers: with HCl 50% homogenized with urine to obtain pH < 2, and without HCl. Each container was divided again into aliquots for immediate measurement or freezing. One aliquot with acid (group 1) and another without acid (group 2) were sent immediately to the laboratory for testing (HPLC), while the other two aliquots, one with acid (group 3) and another without it (group 4) were frozen for 3 months at - 20 °C. Bland-Altman's test was used to analyze inter-assay agreement between measurements. RESULTS: A total of 15 individuals were included (mean age 27.5 ± 5.9 years, 8 male and 14 white). No difference was observed on mean urinary metanephrine/creatinine ratio between groups: group 1: 0.23 ± 0.11, group 2: 0.22 ± 0.07, group 3: 0.25 ± 0.13, group 4: 0.25 ± 0.15 mg/g creatinine; P > 0.05 for all the comparisons). Bland-Altman's analysis showed agreement between the standard method (group 1) and the experimental method (group 4). CONCLUSION: Measurement of urinary metanephrines by HPLC method is not influenced by sample acidification nor freezing at - 20 °C for 3 months.

Comparison of insulin degludec with insulin detemir in type 1 diabetes: a 1-year treat-to-target trial.

The long-term safety and tolerability of insulin degludec (IDeg) was compared with that of insulin detemir (IDet), as basal treatment in participants with type 1 diabetes mellitus (T1DM). In the present multinational, 26-week core + 26-week extension, controlled, open-label, parallel-group trial, adults with T1DM were randomized to IDeg or IDet as basal insulin treatment combined with meal-time bolus insulin aspart. IDeg was administered once daily, whilst IDet was administered once or twice daily depending on patients' glycaemic control. After 1 year, IDeg provided a 33% lower rate of nocturnal hypoglycaemia compared with IDet: estimated rate ratio (IDeg : IDet) 0.67 [95% confidence interval (CI) 0.51; 0.88]; p < 0.05. IDeg improved glycated haemoglobin after 1 year of treatment, similarly to IDet, but IDeg also provided a significantly greater reduction in fasting plasma glucose compared with IDet: estimated difference (IDeg - IDet) -1.11 (95% CI -1.83; -0.40) mmol/l; p < 0.05. The present study confirmed the long-term safety and tolerability profile of IDeg in patients with T1DM. IDeg provided a lower risk of nocturnal confirmed hypoglycaemia than IDet.

Insulin lispro low mixture twice daily versus basal insulin glargine once daily and prandial insulin lispro once daily in patients with type 2 diabetes requiring insulin intensification: a randomized phase IV trial.

AIMS: To compare the efficacy and safety of two insulin intensification strategies in patients with type 2 diabetes inadequately controlled on basal insulin glargine with metformin and/or pioglitazone. METHODS: A multinational, randomized, open-label trial that compared insulin lispro low mixture (LM25; n = 236) twice daily with a basal-prandial regimen of insulin glargine once daily and insulin lispro once daily (IGL; n = 240) over 24 weeks in patients with HbA1c 7.5-10.5% and fasting plasma glucose ≤ 6.7 mmol/l. The primary objective was to assess non-inferiority [per-protocol (PP) population], and then superiority [intention-to-treat (ITT) population], of LM25 versus IGL according to change in HbA1c after 24 weeks (non-inferiority margin 0.4%, two-sided significance level 0.05). RESULTS: Estimated change [least squares (LS) mean (95% CI)] in HbA1c after 24 weeks: -1.30 (-1.44, -1.16)% with LM25 and -1.08 (-1.22, -0.94)% with IGL. Non-inferiority was shown [LS mean (95% CI) HbA1c treatment difference -0.21 (-0.38, -0.04) (PP population)]; gated superiority assessment showed a statistically significant advantage for LM25 (p = 0.010; ITT population). Mean blood glucose, glycaemic variability, overall tolerability and hypoglycaemic episodes per patient-year did not show significant differences between treatments during the study. CONCLUSIONS: In patients with type 2 diabetes inadequately controlled on once-daily basal insulin glargine and metformin and/or pioglitazone, intensification with LM25 was superior to a basal-prandial approach in terms of reduction in HbA1c after 24 weeks and did not increase hypoglycaemia episodes.

Efficacy and safety of insulin degludec given as part of basal-bolus treatment with mealtime insulin aspart in type 1 diabetes: a 26-week randomized, open-label, treat-to-target non-inferiority trial.

AIMS: The efficacy and safety of insulin degludec (IDeg) was compared with insulin detemir (IDet), both administered once daily (OD) as basal treatment in participants with type 1 diabetes mellitus (T1DM). The primary outcome was non-inferiority of IDeg to IDet in glycated haemoglobin (HbA1c) reduction after 26 weeks. METHODS: This multinational, 26-week, controlled, open-label, parallel-group trial randomized adults with T1DM to IDeg or IDet as OD basal insulin treatment combined with mealtime bolus insulin aspart (IAsp). Participants with T1DM treated with any basal-bolus insulin regimen for ≥ 12 months prior to the trial, a mean HbA1c ≤ 10.0% (85.8 mmol/mol) and body mass index (BMI) ≤ 35.0 kg/m(2) at screening participated in the trial (IDeg: N = 302; IDet: N = 153). RESULTS: After 26 weeks, HbA1c decreased 0.73% (8.0 mmol/mol) (IDeg) and 0.65% (7.1 mmol/mol) (IDet) [estimated treatment difference (ETD) IDeg-IDet: -0.09% (-0.23; 0.05)95% CI (-10.0 mmol/mol [-2.6; 0.6]95% CI ); confirming non-inferiority]. Mean fasting plasma glucose improved in both groups, and was lower with IDeg than IDet [ETD IDeg-IDet: -1.66 mmol/l (-2.37; -0.95)95% CI , p < 0.0001]. The rate of confirmed hypoglycaemia was similar with IDeg and IDet [45.83 vs. 45.69 episodes per patient-year of exposure (PYE); estimated rate ratio (RR) IDeg/IDet: 0.98 (0.80; 1.20)95% CI , p = 0.86]. The rate of nocturnal confirmed hypoglycaemia was lower with IDeg than IDet [4.14 vs. 5.93 episodes per PYE; RR IDeg/IDet: 0.66 (0.49; 0.88)95% CI , p = 0.0049]. Adverse event profiles were similar between groups. CONCLUSION: IDeg administered OD in basal-bolus therapy effectively improved long-term glycaemic control in participants with T1DM with a lower risk of nocturnal confirmed hypoglycaemia than IDet.

Long-term 4-year safety of saxagliptin in drug-naive and metformin-treated patients with Type 2 diabetes.

AIMS: To evaluate the safety of saxagliptin ± metformin over 4 years in patients with Type 2 diabetes mellitus. METHODS: Drug-naive (n = 401; study 11) or metformin-treated (n = 743; study 14) adults with HbA(1c) of 53-86 mmol/mol (7.0-10%) were enrolled in two randomized, placebo-controlled, double-blind trials of saxagliptin 2.5, 5 or 10 mg/day. Patients rescued during or completing 24 weeks of treatment could continue in a 42-month long-term blinded phase, for which the primary goal was assessment of safety and tolerability. Between-group efficacy was not evaluated in the long-term phase of study 11. Time to rescue or discontinuation because of inadequate glycaemic control, change from baseline in HbA(1c) and percentages of patients achieving HbA(1c) < 53 mmol/mol (< 7.0%) were assessed in study 14. RESULTS: No new safety findings were noted during the long-term phase. Most adverse events were mild or moderate, with slightly greater frequency of upper respiratory infections with saxagliptin. Hypoglycaemic event rates were similar with saxagliptin and placebo. In study 14, time to rescue or discontinuation because of inadequate glycaemic control was longer with saxagliptin plus metformin than for placebo plus metformin. From baseline to week 154, HbA(1c) decreased with saxagliptin but increased with placebo. CONCLUSION: Saxagliptin monotherapy or add-on to metformin is generally safe and well tolerated, with no increased risk of hypoglycaemia, for up to 4 years.

Impact of adjuvant chemotherapy in completely resected stage IIIA non-small cell lung cancer.

AIM: Outcome of patients with locally advanced non-small-cell lung cancer (NCSLC) is generally poor, with five-year survival rate of only 23%, when patients are treated with surgery only. The presentation of positive adjuvant therapy trials in NSCLC has changed clinical practice, doubling the number of patients with completely resected NSCLC referred for adjuvant chemotherapy since 2004. Furthermore, few large studies described a large number of stage III patients in non-Asiatic patients and they showed controversial results about survival in completely resected stage IIIA NSCLC. The objective of this study was to evaluate the impact of adjuvant chemotherapy in completely resected stage IIIA NCSLC, administered on a routine basis, outside clinical trials. METHODS: This is a retrospective study of patients with stage IIIA NCSLC treated between 1990 and 2008, and included in a continuous, consecutive database. Inclusion criteria were: age >18 years, complete surgical resection, and pathologically confirmed as stage IIIA. The following clinical data were obtained: age, gender, performance status, histological type, chemotherapy regimens, status at last follow-up and hospital where the treatment occurred. Kaplan-Meier's method was used to determine actuarial survival. Differences in survival were determined by Breslow and log rank analyses. RESULTS: According to these inclusion criteria, 415 patients were considered for the present study. The median follow-up time of all patients was 38.2 months. The adjuvant chemotherapeutic treatment affected survival significantly (P <0.001). Also the type of chemotherapeutic treatment affected survival (P ≤0.001). CONCLUSION: Cisplatin-based adjuvant chemotherapy was beneficial in patients who had a completed resected stage IIIA carcinoma.

HbA(1c) measurement for the diagnosis of diabetes: is it enough?

AIM: To analyse the performance of HbA(1c) in diagnosing Type 2 diabetes based on fasting plasma glucose and/or 2-h plasma glucose measurements after a 75-g oral glucose tolerance test. METHODS: This is a study of diagnostic test accuracy in individuals referred to the Clinical Pathology Department for oral glucose tolerance testing. After fasting overnight, HbA(1c), fasting plasma glucose and 2-h plasma glucose were measured. The receiver operating characteristic curve was used to evaluate the diagnostic performance of HbA(1c). RESULTS: Four hundred and ninety-eight subjects (195 male, mean age 56 years) were enrolled and 115 (23.1%) were diagnosed with diabetes according to glucose-based methods and only 56 (11.2%) individuals were identified by HbA(1c) ≥ 6.5% (48 mmol/mol) (sensitivity 20.9%, specificity 95.3%). There is poor agreement between the newly recommended criterion and the current glucose-based diagnostic criteria (κ = 0.217; P < 0.001), probably because the diagnostic methods identify different populations of patients. Adding a glucose-based method into an algorithm, as proposed by the UK Department of Health, improved HbA(1c) performance. CONCLUSIONS: HbA(1c) ≥ 6.5% (48 mmol/mol) showed limited sensitivity to diabetes diagnosis, although with high specificity. The results suggest that this cut-off point would not be enough to diagnose diabetes. Its use as the sole diabetes diagnostic test should be interpreted with caution to assure the correct classification of diabetic individuals.

Hypertension is the metabolic syndrome component most strongly associated with microvascular complications and coronary artery calcification in Type 1 diabetes.

AIM: To evaluate the association of metabolic syndrome (MetS) and its individual components with microvascular complications and coronary artery calcification (CAC) in patients with Type 1 diabetes. MATERIAL/SUBJECTS AND METHODS: Cross-sectional study included 261 patients with Type 1 diabetes. Patients were assessed regarding the presence of MetS according to National Cholesterol Education Program (NCEP) criteria. CAC score was measured in a subset of 100 patients without known cardiovascular disease. RESULTS: The prevalence of MetS was 13.4% according to the NCEP criteria. Microvascular complications and CAC were more frequent in patients with MetS. In a multiple logistic regression analysis, MetS remained associated with nephropathy [OR: 6.33 (95% CI 2.54-15.77), p<0.001], but not with retinopathy and CAC. Among the MetS components, hypertension was associated with presence of retinopathy [OR: 4.04 (95% CI 1.65- 9.90), p=0.002], nephropathy [OR: 5.92 (95% CI 2.42-14.4), p<0.001] and CAC [OR: 2.97 (95% CI 1.06-8.30), p=0.03]. CONCLUSIONS: Hypertension was the only MetS component associated with retinopathy, nephropathy and the presence of CAC. Hypertension was better associated with CAC than MetS itself.

Plasminogen activator and collagenase production by cultured capillary endothelial cells.

Cultured bovine capillary endothelial (BCE) cells produce low levels of collagenolytic activity and significant amounts of the serine protease plasminogen activator (PA). When grown in the presence of nanomolar quantities of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), BCE cells produced 5-15 times more collagenolytic activity and 2-10 times more PA than untreated cells. The enhanced production of these enzymes was dependent on the dose of TPA used, with maximal response at 10(-7) to 10(-8) M. Phorbol didecanoate (PDD), an analog of TPA which is an active tumor promoter, also increased protease production. 4-O-methyl-TPA and 4alpha-PDD, two analogs of TPA which are inactive as tumor promoters, had no effect on protease production. Increased PA and collagenase activities were detected within 7.5 and 19 h, respectively, after the addition of TPA. The TPA-stimulated BCE cells synthesized a urokinase-type PA and a typical vertebrate collagenase. BCE cells were compared with bovine aortic endothelial (BAE) cells and bovine embryonic skin (BES) fibroblasts with respect to their production of protease in response to TPA. Under normal growth conditions, low levels of collagenolyic activity were detected in the culture fluids from BCE, BAE, and BES cells. BCE cells produced 5-13 times the basal levels of collagenolytic activity in response to TPA, whereas BAE cells and BES fibroblasts showed a minimal response to TPA. Both BCE and BAE cells exhibited relatively high basal levels of PA, the production of which was stimulated approximately threefold by the addition of TPA. The observation that BCE cells and not BAE cells produced high levels of both PA and collagenase activities in response to TPA demonstrates a significant difference between these two types of endothelial cells and suggests that the enhanced detectable activities are a property unique to bovine capillary and microvessel and endothelial cells.

Alogliptin added to insulin therapy in patients with type 2 diabetes reduces HbA(1C) without causing weight gain or increased hypoglycaemia.

AIMS: To assess the efficacy and safety of alogliptin added to insulin in patients with type 2 diabetes inadequately controlled with insulin alone or combined with metformin. METHODS: In this 26-week, double-blind, placebo-controlled study, 390 patients were randomized to receive alogliptin 12.5 mg (n = 131), alogliptin 25 mg (n = 129) or placebo (n = 130) once daily, as add-on to stable insulin therapy with or without metformin. The primary endpoint was change in haemoglobin A(1C) (HbA(1C)) at week 26. RESULTS: At week 26, mean HbA(1C) changes from the mean baseline value of 9.3% were significantly greater for alogliptin 12.5 mg (-0.63 +/- 0.08%) and alogliptin 25 mg (-0.71 +/- 0.08%) than placebo (-0.13 +/- 0.08%; p < 0.001). Significantly greater proportions of patients receiving alogliptin 12.5 or 25 mg than placebo had HbA(1C) decreases of > or =0.5, > or =1.0 and > or =1.5%. Insulin doses remained unchanged, and there were no differences in the proportions of patients experiencing hypoglycaemia among placebo (24%), alogliptin 12.5 mg (27%) and alogliptin 25 mg (27%). Mean weight increases from baseline at week 26 were similar for placebo (0.6 +/- 0.2 kg), alogliptin 12.5 mg (0.7 +/- 0.2 kg) and alogliptin 25 mg (0.6 +/- 0.2 kg). Incidences of overall adverse events, and of gastrointestinal, dermatological and infection-related events, were similar among groups. CONCLUSIONS: Adding alogliptin to previous insulin therapy (with or without metformin) significantly improved glycaemic control in patients with type 2 diabetes inadequately controlled on insulin, without causing weight gain or increasing the incidence of hypoglycaemia. Further studies are warranted to explore the role of alogliptin added to optimized basal insulin regimens.

Degree of catecholamine hypersecretion is the most important determinant of intra-operative hemodynamic outcomes in pheochromocytoma.

Pheochromocytoma resection is often complicated by intra-operative hypertension and post-resection hypotension. Factors associated with these hemodynamic alterations are not well defined. The aim of this study was to analyse the clinical-laboratory features associated with hemodynamic parameters during pheochromocytoma resection. Twenty-seven patients submitted to tumor resection - either open (no.=18) or video laparoscopic - between 1978-2007 were included. Nineteen received pre-operative alpha-blockers. Intra-operative hemodynamic data analysed were: maximum and minimum mean arterial blood pressure (MABP), no. of severe hypertensive (systolic BP >200 mmHg) and hypotensive episodes (MABP <60 mmHg), maximum and minimum heart rate (HR), no. of episodes of tachycardia and bradycardia, need to receive iv intra-operative treatment for hypertension and hypotension and the volume of fluids administered during surgery. Patients were 39.4+/-14.4-yr-old, 66% women. Intra-operative hemodynamic parameters were not different in patients submitted to open or video laparoscopic resection. Maximum intraoperative HR and the percentage of patients with HR>100 beats/min were higher in patients without pre-operative alpha- blocker treatment (no.=8). Pre-operative urinary vanylmandelic acid was positively associated with intra-operative maximum MABP (r=0.535, p=0.047) and with maximum transoperative systolic BP (r=0.805, p=0.016). Pre-operative urinary catecholamine (Pearson correlation r=0.575, p=0.03) and vanylmandelic acid (Pearson correlation r=0.605, p=0.04) levels were associated with maximum intra- operative MABP, adjusted for the presence of pheochromocytoma symptoms, surgical approach and pre-operative alpha-blockers. In conclusion, the degree of pre-operative catecholamine secretion was the most important aspect of transoperative BP control.

Impact of metabolic syndrome and C-reactive protein on outcome after coronary stenting.

Metabolic syndrome (MS) identifies cardiovascular risk; however, there is little information regarding the evolution of patients with MS after stent implantation. The aim of this single-center study is to evaluate the possible association between MS and clinical restenosis, after adjustment for highsensitivity C-reactive protein (hs-CRP) and angiographic predictors of restenosis. In a longitudinal study, 159 patients (89 with and 70 without MS) were studied. Criteria for MS were: elevated blood pressure (systolic >or=130 mmHg, diastolic >or=85 mmHg or drug treatment for hypertension; elevated fasting glucose (>100 mg/dl) or drug treatment for elevated glucose; reduced HDL-cholesterol (<40 mg/dl in men and <50 mg/dl in women) or drug treatment for reduced HDL-cholesterol; elevated triglycerides (>or=150 mg/dl) or drug treatment for elevated triglycerides; and obesity (body mass index >28.8 kg/m2). The primary end point was the rate of major adverse clinical events (MACE): cardiovascular death, myocardial infarction, or target lesion revascularization (TLR) during the 12-month follow-up period. The secondary end point was the rate of TLR. MS was neither identified as predictor of MACE [hazard ratio (HR): 0.844; 95% CI: 0.41-1.74; p=0.648], nor TLR (HR: 1.05; 95% CI: 0.44-2.50; p=0.91), even when controlled for hs-CRP levels and angiographic predictors of restenosis. Also, no significant interaction between MS and hs-CRP was found (p=0.135 and p=0.194, for MACE and TLR, respectively). This study shows that patients with MS do not have an additional risk of MACE, even when controlled for angiographic predictors of restenosis and hs-CRP.

A strategy to avoid missed cases in a Brazilian neonatal TSH screening program for congenital hypothyroidism.

BACKGROUND: There have been reports of patients with primary hypothyroidism not identified by TSH measurement due to a presumably delayed rise in serum TSH. However, there are no data on the incidence of false negative results in neonatal screening programs employing primary TSH assay for diagnosis. AIM: To investigate the incidence of false negative results in a neonatal screening program using the primary TSH approach and evaluate a strategy to avoid misdiagnosis. INFANTS AND METHODS: 190 newborns, with initial TSH > 15.0 IU/l and < 20.0 IU/l (screening cutoff). These infants were submitted to a second TSH measurement around 30 days after the first screening. RESULTS: Thirty days after the first screening, four of the 190 infants (2.1%) remained with TSH levels around the cut-off screening level or higher. Of these four patients, three had an absence of thyroid gland on ultrasonography, with a final diagnosis of dysgenesis. The fourth patient had a normal shaped gland in its usual location by ultrasonography, with an outcome of transitory congenital hypothyroidism. CONCLUSIONS: The incidence of missed congenital hypothyroidism diagnoses in this neonatal screening program based on the TSH approach was low and acceptable. Nevertheless, with the proposed strategy, the risk of false negative results can be reduced without significant impact on the overall cost of the screening program.

Blood pressure means rather than nocturnal dipping pattern are related to complications in Type 2 diabetic patients.

AIM: To determine whether systolic and diastolic blood pressure (BP) means, during ambulatory BP monitoring (ABPM), are more strongly correlated with microvascular complications and echocardiographic structural alterations than night-time/daytime (N/D) BP ratio. METHODS: A cross-sectional study was conducted in 270 Type 2 diabetes mellitus (DM) outpatients who underwent clinical and laboratory investigations, urinary albumin excretion rate (UAER) determination, echocardiography, office and 24-h ABPM (Spacelabs 90207). RESULTS: UAER, after multivariate adjustments, was associated with office BP (systolic: R(2)(a) 0.162, P < 0.001; diastolic: R(2)(a) 0.124, P < 0.001) and ABPM (24-h systolic: R(2)(a) 0.195, P < 0.001; 24-h diastolic: R(2)(a) 0.197, P < 0.001) but not with N/D BP ratios (systolic: R(2)(a) 0.062, P = 0.080; diastolic: R(2)(a) 0.063, P = 0.069). Similar results were observed for echocardiographic parameters. The presence of retinopathy was associated only with night-time BP values [systolic means: odds ratio (OR) 1.13, 95% confidence interval (CI) 1.03-1.24 and diastolic means: OR 1.21, CI 1.04-1.40 and N/D diastolic BP ratio > 0.90, OR 3.21, CI 1.65-6.25]. CONCLUSIONS: UAER and echocardiographic structural alterations had more consistent correlations of a greater magnitude with systolic BP means than with N/D BP ratios. The nocturnal BP values appear to be more relevant for diabetic retinopathy. BP measurement in patients with Type 2 DM should take into account the 24-h period rather than focusing on a specific time span of BP homeostasis.

Proliferative diabetic retinopathy is associated with microalbuminuria in patients with type 2 diabetes.

Diabetic retinopathy is one of the leading causes of blindness in working-age individuals. Diabetic patients with proteinuria or those on dialysis usually present severe forms of diabetic retinopathy, but the association of diabetic retinopathy with early stages of diabetic nephropathy has not been entirely established. A cross-sectional study was conducted on 1214 type 2 diabetic patients to determine whether microalbuminuria is associated with proliferative diabetic retinopathy in these patients. Patients were evaluated by direct and indirect ophthalmoscopy and grouped according to the presence or absence of proliferative diabetic retinopathy. The agreement of diabetic retinopathy classification performed by ophthalmoscopy and by stereoscopic color fundus photographs was 95.1% (kappa = 0.735; P < 0.001). Demographic information, smoking history, anthropometric and blood pressure measurements, glycemic and lipid profile, and urinary albumin were evaluated. On multiple regression analysis, diabetic nephropathy (OR = 5.18, 95% CI = 2.91-9.22, P < 0.001), insulin use (OR = 2.52, 95% CI = 1.47-4.31, P = 0.001) and diabetes duration (OR = 1.04, 95% CI = 1.01-1.07, P = 0.011) were positively associated with proliferative diabetic retinopathy, and body mass index (OR = 0.90, 95% CI = 0.86-0.96, P < 0.001) was negatively associated with it. When patients with macroalbuminuria and on dialysis were excluded, microalbuminuria (OR = 3.3, 95% CI = 1.56-6.98, P = 0.002) remained associated with proliferative diabetic retinopathy. Therefore, type 2 diabetic patients with proliferative diabetic retinopathy more often presented renal involvement, including urinary albumin excretion within the microalbuminuria range. Therefore, all patients with proliferative diabetic retinopathy should undergo an evaluation of renal function including urinary albumin measurements.

Effects of modulation of basic fibroblast growth factor on tumor growth in vivo.

BACKGROUND: Recombinant human basic fibroblast growth factor (rHu-bFGF) is known to stimulate proliferation in some tumor cells and to modulate tumor vascularization. PURPOSE: The purpose of this study was to examine the possible role of this agent in the development of tumors. The study was designed to determine the effects of modulating bFGF activity in vivo in tumor models from cell lines with different responses to bFGF and with different content and receptor levels of bFGF. METHODS: Two tumor cell lines (human DLD-2 colon carcinoma and rat C6 glioma) were characterized for bFGF content and bFGF receptor levels by Western blot analysis in cultured cells and by studies of [125I]rHu-bFGF binding to sections from xenografts grown in nude mice. Tumor cell proliferation was monitored after treatment with rHu-bFGF or the DG2 or DE6 IgG monoclonal antibody to rHu-bFGF in culture and in vivo. RESULTS: C6 cells exhibited 7800 high-affinity receptors for rHu-bFGF per cell (dissociation constant [Kd] = 46 pM), while DLD-2 cells lacked high-affinity receptors. rHu-bFGF stimulated [3H]thymidine uptake by C6 cells, but the addition of DG2 IgG prevented this stimulation; rHu-bFGF had no effect on [3H]thymidine incorporation by DLD-2 cells. C6 cells had higher levels of immunoreactive bFGF than did DLD-2 cells. The xenografts from both cell lines exhibited high-affinity [125I]rHu-bFGF binding that was concentrated on vascular-like structures. rHu-bFGF at a dosage of 0.25 mg/kg given intraperitoneally daily for 18 days caused a twofold increase in DLD-2 tumor weight but had little effect on the growth of C6 xenografts. In contrast, daily intravenous injections of DG2 IgG given to mice had no effect on DLD-2 tumor growth but reduced growth of C6 tumors by approximately 30%--a statistically significant difference. CONCLUSIONS: The addition of exogenous rHu-bFGF or of a neutralizing antibody resulted in significant alterations in tumor growth in vivo, which were specific for tumor type and bFGF characteristics. While some of these effects may be mediated by the bFGF-responsive endothelial cells of the tumor vasculature (DLD-2 colon carcinoma), others may result from inhibition of bFGF-dependent tumor cell proliferation (C6 glioma). IMPLICATIONS: Studies that measure tumor blood flow are necessary to confirm that these effects are mediated by changes in tumor vasculature.

Activity of DMP 840, a new bis-naphthalimide, on primary human tumor colony-forming units.

BACKGROUND: DMP 840 ((R,R)-2,2'-[1,2-ethanediylbis[imino(1-methyl-2,1-ethanediyl)]]- bis[5-nitro-1H-benz[de]-iso[quinoline-1,3(2H)-dione]dimethane- sulfonate; NSC-D640430) is one in a series of bis-naphthalimides that binds DNA with high affinity and has sequence specificity to multiple G and C bases. It is also a potent inhibitor of RNA synthesis. DMP 840 has been selected for clinical evaluation on the basis of a broad spectrum of activity (including cures) in human tumors in murine models. PURPOSE: We evaluated DMP 840 in a human tumor clonogenic assay to estimate what plasma concentrations may be necessary for clinical cytotoxic activity and to determine what types of tumors potentially might be primary targets for initial phase II studies. METHODS: A soft-agar cloning system assay was used to determine the in vitro effects of DMP 840 against cells from biopsy specimens of colorectal, breast, lung ovarian, renal cell, stomach, and bladder cancers and from other tumor types. A total of 260 human tumor specimens were exposed continuously during the assay to DMP 840; 103 were assessable (20 colonies or more on control plates and 30% or less survival for the positive control). An in vitro response was defined as at least a 50% decrease in tumor colony formation resulting from drug exposure compared with controls. RESULTS: In vitro responses were seen in 10% (one of 10), 54% (55 of 101), 80% (82 of 103), and 89% (82 of 92) of specimens tested at 0.01, 0.1, 1.0, and 10.0 micrograms/mL of DMP 840, respectively. At a concentration of 0.1 microgram/mL, specific activity was seen against melanoma (80%) and against renal cell (80%), ovarian (63%), breast (54%), non-small-cell lung (42%), and colorectal cancers (33%). DMP 840 demonstrated activity in tumor specimens resistant in vitro to methotrexate (88%), doxorubicin (58%), platinum (57%), cyclophosphamide (53%), vinblastine (53%), etoposide (53%), fluorouracil (37%), and paclitaxel (36%). CONCLUSIONS: At in vitro concentrations of 0.1 microgram/mL as a continuous exposure, DMP 840 has activity against a variety of human tumors, including a subgroup resistant in vitro to standard antineoplastic agents. IMPLICATIONS: Further clinical development of DMP 840 is warranted.

Plasminogen activator and inhibitor activity in human glioma cells and modulation by sodium butyrate.

The activity of the serine protease plasminogen activator (PA), which correlates with tumorigenicity and metastatic capacity, was examined using the 125I-labeled fibrin plate assay in cell extracts from four human glioma lines as a function of growth in vitro. Cell-associated inhibitory activity to plasmin and urokinase-type PA was also measured concurrently. The relative PA activities differed markedly among the lines, whereas inhibitory activities did not. Two lines, SNB-19 and SNB-75, exhibited maximal PA activities (1-6 m Plough units/micrograms protein) as cultures approached confluence, whereas two other lines, SNB-56 and SNB-78, expressed low PA activity at all times (less than 0.2 m Plough units/micrograms protein). The PA of SNB-19 cell extracts was predominantly urokinase-type PA. In addition to having the highest PA levels, SNB-19 and SNB-75 were the most clonogenic in soft agar and tumorigenic in nude mice. In contrast, SNB-56 and SNB-78 were poorly clonogenic in soft agar and were not tumorigenic in nude mice. Measured directly, inhibitory activities to plasmin, urokinase-type PA, and tissue-type PA were detected in SNB-19 (high PA) and SNB-56 (low PA) cell extracts. However, there were no qualitative or quantitative differences in inhibitor effects between SNB-19 and SNB-56 suggesting that the differences in PA activity between these lines resulted from changes in PA activity and were not due to differential plasminogen activator inhibitor effects. The ability of the differentiating agent sodium butyrate (NaB) to modulate total PA activity was also examined. Peak SNB-19 cell PA activity was decreased in a concentration (Ki, 0.75 mM) and time-dependent manner by the addition of nontoxic amounts of NaB. The dose-dependent decrease in PA activity induced by NaB was most likely due to an effect on PA itself, since the action of inhibitor on urokinase was unchanged in response to NaB. These results suggest that net cellular PA activity in glioma cells is a balance between relative PA activity and inhibitor(s) effects and that this balance can be modulated by sodium butyrate.

Immunoconjugates containing novel maytansinoids: promising anticancer drugs.

The potential of immunoconjugates of cytotoxic drugs for the treatment of cancer has not yet been realized owing to the difficulty of delivering therapeutic concentrations of these drugs to the target cells. In an effort to overcome this problem we have synthesized maytansinoids that have 100- to 1000-fold higher cytotoxic potency than clinically used anticancer drugs. These maytansinoids are linked to antibodies via disulfide bonds, which ensures the release of fully active drug inside the cells. The conjugates show high antigen-specific cytotoxicity for cultured human cancer cells (50% inhibiting concentration, 10 to 40 pM), low systemic toxicity in mice, and good pharmacokinetic behavior.