Interspecies differences in the kinetic properties of deoxycytidine kinase elucidate the poor utility of a phase I pharmacologically directed dose-escalation concept for 2-chloro-2′-deoxyadenosine

2-Chloro-2-deoxyadenosine (CdA, Cladribine), is a purine antimetabolite currently under investigation in phase II clinical trials for the treatment of lymphoid malignancies. Significant differences in CdA toxicity between mice and humans were observed during phase I clinical evaluation. For the elucidation of interspecies differences in drug toxicity the pharmacokinetics of CdA after subcutaneous injection and the kinetic properties of the CdA-phosphorylating enzyme, deoxycytidine kinase (dCK), were compared in mice and humans. The ratio of the dose lethal to 10% of mice (LD₁₀) to the maximum tolerated dose (MTD) in humans was 50 and the ratio of the area under the curve obtained at approximately one-half the LD₁₀ (AUC_{approx. one-half the LD 10})/AUC_MTD was 49. A significant interspecies difference was observed in the kinetic properties of dCK, the main CdA-activating enzyme. With CdA as a substrate, the Michaelis constant (K _m) of dCK in crude extracts of mouse thymus was 10 times higher than that in human thymus. An approximately 9-fold interspecies difference in maximum velocity (V_max)/K _m indicated a higher efficiency of dCK for CdA in humans than in mice. The peak plasma concentration was 210 times higher and exceeded theK _m in mice. Initial and terminal half-lives were approximately 7 times shorter in mice and trough levels were similar in mice and humans. Thus, the differences in AUCs at equitoxic doses are largely explained by differences in the target enzyme properties and the pharmacokinetic pattern. The observed lower tolerance for CdA in humans as compared with mice confirms the view that antimetabolites may not be good candidates for pharmacokinetically guided dose-escalation schemes unless detailed information on interspecies variability in drug bioactivation is available.     

Biomarkers of styrene exposure in lamination workers: levels of 06-guanine DNA adducts, DNA strand breaks and mutant frequencies in the hypoxanthine guanine phosphoribosyltransferase gene in T-lymphocytes

Occupational exposure to styrene was studied in nine workersof a hand lamination plant in Bohemia. Personal dosimeters wereused to monitor the styrene workplace exposure, and the levelsof styrene in blood and mandelic acid in urine were measured.Blood samples were taken at four occasions during a 7 monthperiod to determine styrene-specific 0⁶-guanine DNA adductsin lymphocytes and granulocytes, DNA strand breaks and hypoxanthineguanine phosphoribosyltransferase (HPRT) mutant frequency inT-lymphocytes. Seven administrative employees in the same factory(factory controls) and eight persons in a research laboratory(laboratory controls) were used as referents. DNA adduct levelsdetermined by the ³²P-postlabelling method in lymphocytes oflamina-tors were remarkably constant and significantly higher(P < 0.0001) than in factory controls at all four samplingtimes. HPRT mutant frequencies (MF) measured by the T-cell cloningassay were higher in the laminators (17.5 x10^–6, groupmean) than in the factory controls (15.7x10^–6, group mean)at three of the four sampling times, but the differences werenot statistically significant. However, a statistically significant(P = 0.021) difference between MF in the laminators (18.0 x10^–6,group mean) and laboratory controls (11.8 xl0^–6, groupmean) was observed at sampling time 4 (the only sampling timewhen this latter group was studied). This result indicates thatstyrene exposure may induce gene mutation in T-cells in vivo.DNA strand breaks were studied by the ‘Comet assay’at the fourth sampling time. The laminators were found to havesignificantly higher levels of DNA strand breaks than the factorycontrols (P = 0.032 for tail length, TL; P = 0.007 for percentageof DNA in tail, T%; and P = 0.020 for tail moment, TM). A statisticallysignificant correlation was also found between the levels oflymphocyte DNA adducts and all three DNA strand break parameters(TL P = 0.046; T% P = 0.026 and TM P = 0.034). On the contrary,no significant correlations were found between DNA adduct levelsand the HPRT mutant frequencies or between the mutant frequenciesand DNA strand breaks. Taken together, these results add furthersupport to the genotoxic and possibly mutagenic effects of styreneexposure in vivo. However, no simple quantitative relationshipseems to exist between the levels of styrene-induced DNA damageand frequency of HPRT mutation in T-lymphocytes.     

Presence of functionally active β-adrenoceptors in rat mast cells

Summary The correlation between the binding of a -adrenoceptor antagonist, (–)[³H]-dihydroalprenolol (DHAP), and the adrenergic inhibition of histamine release by acetylcholine and by compound 48/80 was studied with isolated purified rat mast cells and in rat mast cell crude membrane fractions.Acetylcholine-evoked histamine release was inhibited by catecholamines, in the order isoprenaline > adrenaline > noradrenaline. Pretreatment of cells with (–)alprenolol antagonized the inhibitory effect of isoprenaline on acetylcholine-induced histamine release.40/80-evoked histamine release was bocked by isoprenaline at significantly higher concentrations than those required to inhibit cholinergic histamine release. The inhibitory effect of isoprenaline was equally antagonized by preincubating mast cells with (–)alprenolol.Specific binding sites for DHAP have been demonstrated in rat mast cell membranes. The specific binding of DHAP was inhibited by adrenoceptor agonists and antagonists according to the stereospecificity of these compounds.A close correlation between the binding-inhibitory potency of various adrenergic compounds and the data obtained in the pharmacological experiments was found, thus indicating the presence of -adrenoceptors in rat mast cells.     

Radiotherapy with or without mitomycin c in the treatment of locally advanced head and neck cancer: results of the IAEA multicentre randomised trial.

BACKGROUND AND PURPOSE: Single agent mitomycin c (MMC) has been shown to improve the outcome of radiotherapy in single institution trials. In order to confirm these findings in a broader worldwide setting, the International Atomic Energy Agency (IAEA) initiated a multicentre trial randomising between radiotherapy alone versus radiotherapy plus MMC. MATERIAL AND METHODS: Patients with advanced head and neck cancer were treated with primary curative radiotherapy (66 Gy in 33 fractions with five fractions per week) +/-a single injection (15 mg/m(2)) of MMC at the end of the first week of radiotherapy. Stratification parameters were tumour localization, T-stage, N-stage, and institution. A total of 558 patients were recruited in the trial from February 1996 to December 1999. Insufficient accrual and reporting led to the exclusion of three centres. The final study population consisted of 478 patients from seven centres. Patients had stage III (n=223) or stage IV (n=255) squamous cell carcinoma of the oral cavity (n=230), oropharynx (n=140), hypopharynx (n=65) or larynx (n=43). Prognostic factors like age, gender, site, size, differentiation and stage were well balanced between the two arms. RESULTS: The haematological side effects of MMC were very modest (<5% grade 3-4) and did not require any specific interventions. Furthermore, MMC did not enhance the incidence or severity of acute and late radiation side effects. Confluent mucositis and dry skin desquamation was common, occurring in 56% and 62% of patients, respectively. The overall 3-year primary locoregional tumour control, disease-specific and overall survival rates were 19, 36 and 30%, respectively. Gender, haemoglobin drop, tumour site, tumour and nodal stage were significant parameters for loco-regional tumour control. There was no significant effect of MMC on locoregional control or survival, except for the 161 N0 patients, where MMC resulted in a better loco-regional control (3-year estimate 16% vs. 29%, P=0.01). CONCLUSIONS: The study did not show any major influence of MMC on loco-regional tumour control, survival or morbidity after primary radiotherapy in stage III-IV head and neck cancer except in N0 patients where loco-regional control was significantly improved.     

Mutations in the tyrosine kinase domain of the epidermal growth factor receptor in non-small cell lung cancer.

We evaluated somatic genetic alterations in the kinase domain of the EGFR gene in the tumors of 219 non-small cell lung cancer patients of primarily Caucasian and African American origins. We identified 26 patients (12%) whose tumors had a mutation in the EGFR gene, and 11 (5%) patients carried novel genomic variations consistent with germ-line polymorphisms. All but one mutation were identified in Caucasian patients affected with adenocarcinoma. EGFR mutations were more frequent in women and in nonsmokers, but a significant portion of the affected patients were men (12 of 26) and current or past smokers accounted for half of the patients affected (13 of 26). Screening subjects with EGFR mutations may identify patients whose tumors could respond to targeted therapy using tyrosine kinase inhibitors.     

Artificial Anisotropy in Ge2Sb2Te5 Thin Films after Femtosecond Laser Irradiation

Ge₂Sb₂Te₅ (GST225) looks to be a promising material for rewritable memory devices due to its relatively easy processing and high optical and electrophysical contrast for the crystalline and amorphous phases. In the present work, we combined the possibilities of crystallization and anisotropic structures fabrication using femtosecond laser treatment at the 1250 nm wavelength of 200 nm thin amorphous GST225 films on silicon oxide/silicon substrates. A raster treatment mode and photoexcited surface plasmon polariton generation allowed us to produce mutually orthogonal periodic structures, such as scanline tracks (the period is 120 ± 10 μm) and laser-induced gratings (the period is 1100 ± 50 nm), respectively. Alternating crystalline and amorphous phases at the irradiated surfaces were revealed according to Raman spectroscopy and optical microscopy studies for both types of structures. Such periodic modulation leads to artificial optical and electrophysical anisotropy. Reflectance spectra in the near infrared range differ for various polarizations of probing light, and this mainly results from the presence of laser-induced periodic surface structures. On the other hand, the scanline tracks cause strong conductivity anisotropy for dc measurements in the temperature range of 200–400 K. The obtained results are promising for designing new GST225-based memory devices in which anisotropy may promote increasing the information recording density.     

Conductive Layers on a Shrinkable PET Film by Flexographic Printing

In this study, the extremely important and difficult topic of flexographic printing on a heat-shrinkable substrate was taken up. Six commercially available, electrically conductive inks based on silver, copper and graphite nanoparticles were selected and tested upon their applicability for printing on the temperature-sensitive PET material. As a printing substrate, the one-direction heat-shrinkable PET film, with a maximum shrinkage of 78%, was selected. All of the examined inks were subjected to the printing process throughout three different anilox line screens. The tested inks, along with the electric paths printed with them, were subjected to various tests. The main parameters were evaluated, such as printability combined with the rheology tests and ink adhesion to the examined PET substrate together with the electrical conductivity before and after the shrinkage.     

Desempenho Diagnóstico da Angiotomografia Computadorizada e da Avaliação Seriada de Troponina Cardíaca Sensível em Pacientes com Dor Torácica e Risco Intermediário para Eventos Cardiovasculares

BackgroundCoronary tomography angiography (CTA) has been mainly used for chest pain evaluation in low-risk patients, and few data exist regarding patients at intermediate risk.ObjectiveTo evaluate the performance of serial measures of sensitive troponin and CTA in intermediate-risk patients.MethodsA total of 100 patients with chest pain, TIMI risk scores of 3 or 4, and negative troponin were prospectively included. All patients underwent CTA and those with coronary stenosis ≥ 50% were referred to invasive coronary angiography. Patients with coronary lesions <50% were discharged and contacted 30 days later by a telephone call to assess clinical outcomes. Outcomes were hospitalization, death, and myocardial infarction at 30 days. The comparison between methods was performed by Kappa agreement test. The performance of troponin measures and CTA for detecting significant coronary lesions and clinical outcomes was calculated. Results were considered statistically significant when p < 0.05.ResultsCoronary stenosis ≥ 50% on CTA was found in 38% of patients and significant coronary lesions on coronary angiography were found in 31 patients. Two clinical events were observed. Kappa agreement analysis showed low agreement between troponin measures and CTA in the detection of significant coronary lesions (kappa = 0.022, p = 0.78). The performance of CTA for detecting significant coronary lesions on coronary angiography or for predicting clinical events at 30 days was better than sensitive troponin measures (accuracy of 91% versus 60%).ConclusionCTA performed better than sensitive troponin measures in the detection of significant coronary disease in patients with chest pain and intermediate risk for cardiovascular events.     

Left ventricular isovolumic relaxation and renin-angiotensin system in the growth restricted fetus

OBJECTIVE: To determine left ventricular isovolumic relaxation time (LV IRT) in normally developing and growth restricted fetuses (FGR) as an indicator of fetal cardiac afterload and neonatal systolic blood pressure. STUDY DESIGN: A prospective longitudinal study in 124 normally developing and 47 growth restricted fetuses (FGR). LV IRT, fetal heart rate (FHR) and umbilical artery pulsatility index (PI) were determined at 2-3 week intervals starting at 22-26 weeks of gestation until delivery. Renin and angiotensin I levels were measured by radioimmunoassay in umbilical venous blood after delivery. Systolic blood pressure was measured at day 1 and day 5 of postnatal life. To evaluate the association between LV IRT, gestational age and FHR, bivariate regression analyses were performed. RESULTS: Mean LV IRT (62+/-8ms) was 29 percent longer in FGR as compared to the normal subset (47+/-6ms) at all gestational ages (p<0.001). Mean postnatal active plasma renin level (7.78+/-S.D. 1.03ng/ml) and postnatal angiotensin I level (4.21+/-0.70ng/ml) in the FGR subset were significantly higher (p<0.001) than in the normal subset (4.81+/-1.04ng/ml, renin and 2.69+/-0.44ng/ml, angiotensin I). There was a significant difference (p<0.01) in systolic blood pressure between the two subsets on postnatal day 1 (FGR 52+/-6mmHg vs. normal 46+/-4mmHg) and day 5 (FGR 76+/-5mmHg vs. normal 60+/-6mmHg). CONCLUSION: Left ventricular isovolumic relaxation time may act as a sensitive index of increased arterial afterload in the growth retarded fetus and may herald raised systolic blood pressure in the early neonatal period.     

Glyburide metabolism by placentas of healthy and gestational diabetics

The aim of this investigation was to determine the metabolism of glyburide (GL) by microsomes prepared from placentas obtained from uncomplicated pregnancies (UP), women with gestational diabetics (GD) on a diabetic diet, and those on a diet and GL. Term placentas were obtained from UP and GD. Crude microsomal fractions were prepared by differential centrifugation and stored at -80 degrees C. The activity of the microsomes in metabolizing glyburide to the trans-4-hydroxycyclohexyl glyburide (THCGL) and cis-3-hydroxycyclohexyl glyburide (CHCGL) was determined and quantified using high-performance liquid chromatography-mass spectrometer (HPLC-MS). The activity of the placental microsomes varied widely between individual placentas in each group. The median values (pmol.mg (-1) P.min (-1)) for the rates of THCGL formation were 0.34, 0.3, and 0.23 for placentas of UP, GD on diet, and GD on GL and a diet, respectively. The median values for CHCGL formation were 0.13 for UP, 0.11 for GD on a diet, and 0.10 (pmol.mg (-1) P.min (-1)) for GD on GL and a diet. A pool of individual microsomal fractions from each group was prepared and its activity revealed the following: greater formation of THCGL in the UP (0.36 +/- 0.10) than GD (0.22 +/- 0.03) ( P = 0.058 for GD on a diet, 0.04 for GD on GL). There was greater formation of CHCGL in UP (0.26 +/- 0.04) than GD (0.12 +/- 0.003) ( P < 0.006). There was no difference in GD on a diet and GD on GL plus diet. We concluded that the apparent differences in the formation of metabolites may be statistically significant, but it is unlikely to be of physiological importance, given the sample size and other experimental factors. Therefore, a more comprehensive investigation is underway.