Background / Aim. MDSCs suppress immune responses via a series of inhibitory mechanisms, which ultimately could lead to tumor growth. B7-H4 expression is significantly associated with poor outcome and promotion of tumor cell proliferation, invasion and migration in patients with various cancers. Data concerning B7-H4 expression in lung cancers, either on tumor or immunological cells, are still sporadically. To estimate and correlate the number of CD14+B7-H4+MDSC in blood and lung tumor microcirculation with clinical stage, histology type of tumor, TNM stadium, nodal status and disease outspread. Methods. 44 lung cancer patients (III and IV clinical stage) and 30 healthy controls. CD14+B7-H4+ MDSC number was estimated by flowcytometry in blood and tumor microcirculation samples of each patient. Results. CD14+B7-H4+MDSC number was significantly higher in patient’s samples comparing to controls. CD14+B7-H4+MDSC was significantly increased in tumor comparing to blood sample of same patient. Clinical stage III patients had increased number of the CD14+B7-H4+ MDSC comparing to stage IV, in both type of samples. According to histology SCLC patients had the highest average CD14+B7-H4+MDSC number, significantly increased comparing to patients with squamous and large cell LC histology in tumor. Tumor size was directly associated with the number of the CD14+B7-H4+MDSC, both in blood and tumor samples. Furthermore, nodal involvement was associated with gradual increase of the CD14+B7-H4+MDSC number, being the highest in the N3 group, again both in blood and tumor samples. Finally, we have detected higher CD14+ B7-H4+MDSC number in the samples of patients without metastases. Conclusion. CD14+B7-H4+MDSC number in LC patients is significantly associated with tumor histology type, lymph node involvement, disease extent degree and tumor size. Concerning their large number in LC tumor microenvironment together with immunosuppressive capacities, CD14+B7-H4+MDSC could represent important tumor promoting factor in LC pathophysiology.

It was reported that novel O,O′-diethyl-(S, S)-ethylenediamine-N,N′-di-2-(3-cyclohexyl) propanoate dihydrochloride (DE-EDCP) displayed in vitro antiproliferative activity on several human and mouse cancer cell lines, which was comparable to that of the prototypical anticancer drug cisplatin. In order to reveal its toxicity profile, acute and repeated-dose toxicity studies were performed in Naval Medical Research Institute (NMRI) Han mice. The intravenous LD50 values of DE-EDCP were found to be 95.3 and 101.3 mg/kg body weight in female and male mice, respectively. In the subacute toxicity study, DE-EDCP was administered intravenously at the doses of 15, 25, and 40 mg/kg/day for a period of 28 days. There were no adverse effects on general condition, growth, feed and water consumption, and hematological parameters. There was a significant increase in urea and alanine aminotransferase in female mice and aspartate aminotransferase and alkaline phosphatase in both genders in 40 mg/kg/day dose-treated group. The histopathological changes confined to the liver and kidney, but in other organs were not found. Satellite group revealed that changes in the kidney and liver were less pronounced, suggesting their reversibility. Interactions with DNA could also be of importance for understanding DE-EDCP toxic side effects. Hyperchromic effect obtained with ultraviolet–visible, suggested electrostatic interactions between DE-EDCP and calf thymus DNA. The toxicity testing of DE-EDCP was conducted to predict human outcomes.

Melanoma, an aggressive skin tumor with high metastatic potential, is associated with high mortality and increasing morbidity. Multiple available chemotherapeutic and immunotherapeutic modalities failed to improve survival in advanced disease, and the search for new agents is ongoing. The aim of this study was to investigate antimelanoma effects of O,O-diethyl-(S,S)-ethylenediamine-N,N′di-2-(3-cyclohexyl) propanoate dihydrochloride (EE), a previously synthesized and characterized organic compound. Mouse melanoma B16 cell viability was assessed using acid phosphatase, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, sulforhodamine B, and lactate dehydrogenase assays. Apoptosis and autophagy were investigated using flow cytometry, fluorescence and electron microscopy, and western blotting. In vivo antitumor potential was assessed in subcutaneous mouse melanoma model after 14 days of treatment with EE. Tumor mass and volume were measured, and RT-PCR was used for investigating the expression of autophagy-related, proapoptotic, and antiapoptotic molecules in tumor tissue. Investigated organic compound exerts significant cytotoxic effect against B16 cells. EE induced apoptosis, as confirmed by phosphatidyl serine externalisation, caspase activation, and ultrastructural features typical for apoptosis seen on fluorescence and electron microscopes. The apoptotic mechanism included prompt disruption of mitochondrial membrane potential and oxidative stress. No autophagy was observed. Antimelanoma action and apoptosis induction were confirmed in vivo, as EE decreased mass and volume of tumors, and increased expression of several proapoptotic genes. EE possesses significant antimelanoma action and causes caspase-dependent apoptosis mediated by mitochondrial damage and reactive oxygen species production. Decrease in tumor growth and increase in expression of proapoptotic genes in tumor tissue suggest that EE warrants further investigation as a candidate agent in treating melanoma.

A series of new (S,S)-ethylenediamine-N,N′-di-2-(3-cyclohexyl)propanoate esters has shown cytotoxic activity towards human leukemic cell lines. The aim of this study was to develop and validate a bioanalytical method for quantification of (S,S)-O,O-diethyl-ethylenediamine-N,N′-di-2-(3-cyclohexyl)propanoate dihydrochlorides (DE-EDCP) and its metabolite, substituted propanoic acid (EDCP), in mouse serum by ultra high-performance liquid chromatography—tandem mass spectrometry (UHPLC—MS/MS). Structural analog, derivative of 1,3-propanediamine, was used as an internal standard (IS). Sample preparation employed protein precipitation by acetonitrile and subsequent centrifugation. Optimal UHPLC separation conditions were set to achieve simultaneous determination of both compounds in a short run time of 6 min. Additionally, the selected reaction monitoring (SRM) mode developed in this method allowed a highly sensitive, accurate, and precise identification of compounds of interest. The lower limit of quantitation (...

BACKGROUND Benzodiazepines have a direct bronchodilatory effect. Methacholine is a non-selective muscarinic receptor agonist causing bronchoconstriction. AIM To examine the effects of inhaled benzodiazepines, modulating bronchoconstriction induced by methacholine in patients with asthma. PATIENTS AND METHODS Twelve patients with well controlled asthma were studied. On the first day, after determining the initial values of pulmonary function, a dose response curve was carried out with progressive doses of methacholine. After the last dose, when at least a 20% drop of the initial forced expiratory volume in the first second (FEV1) was achieved, vital capacity (VC) and FEV1 were measured at 7, 15 and 30 minutes after provocation. On the second day a diazepam aerosol was inhaled by the patients prior to the same protocol with methacholine. RESULTS In the first day of testing, methacholine inhalation (6 mg/mL) led to a significant drop in FEV1 from 2.98 to 1.69 L. On the second day of study, in the same patients, previous inhalation with diazepam reduced the changes of FEV1 after inhalation of methacholine. This parameter decreased from 2.48 to 2.21 L. CONCLUSIONS Inhalation of benzodiazepines reduce bronchoconstriction after a methacholine challenge in patients with asthma.

Background: Benzodiazepines have a direct bronchodilatory effect. Methacholine is a non-selective muscarinic receptor agonist causing bronchoconstriction. Aim: To examine the effects of inhaled benzodiazepines, modulating bronchoconstriction induced by methacholine in patients with asthma. Patients and methods: Twelve patients with well controlled asthma were studied. On the first day, after determining the initial values of pulmonary function, a dose response curve was carried out with progressive doses of methacholine. After the last dose, when at least a 20% drop of the initial forced expiratory volume in the first second (FEV1) was achieved, vital capacity (VC) and FEV1 were measured at 7, 15 and 30 minutes after provocation. On the second day a diazepam aerosol was inhaled by the patients prior to the same protocol with methacholine. Results: In the first day of testing, methacholine inhalation (6 mg/mL) led to a significant drop in FEV1 from 2.98 to 1.69 L. On the second day of study, in the same patients, previous inhalation with diazepam reduced the changes of FEV1 after inhalation of methacholine. This parameter decreased from 2.48 to 2.21 L. Conclusions: Inhalation of benzodiazepines reduce bronchoconstriction after a methacholine challenge in patients with asthma.

The extensive development of radiopharmaceuticals towards early tumour detection and treatment has increased the demand for new ligands with higher tumour selectivity. Research has been done on the potential of the novel O,O′-diethylethylenediamine-N,N′-di-3-propanoate () ligand as a radionuclide vehicle for tumour targeting. Under alkaline conditions, hydrolyses and produces half ester ligand () and diacid ligand (), with characteristic donor atom array N,N,O. Ligand was successfully labelled with 99mTc at pH = 9 by coordination with the octahedral fac-[99mTc(CO)3(H2O)3]+ intermediate, forming the main radioproduct fac-[99mTcL′(CO)3] (Tc1). The 99mTc complex showed a low lipophilic character (log P = 0.48) and low binding affinity to human serum albumin (2.51 ± 0.48%). In vitro stability studies in saline and human plasma, as well as challenge studies with cysteine and histidine, revealed high stability of the complex during 24 h. Biodistribution studies of Tc1 in female C57BL/6 mice bearing B16/F1 melanoma metastases showed significant tumour uptake: 9.81 ± 1.19%ID g−1 in the liver, 5.87 ± 0.54%ID g−1 in the lungs and 3.17 ± 0.33%ID g−1 in the ovary at 30 min post-injection. Favourable physicochemical properties, satisfactory in vitro/in vivo stability and biodistribution profile in the experimental metastatic melanoma model indicate the possible application of the radiolabelled ligand in tumour diagnosis. Copyright © 2015 John Wiley & Sons, Ltd.

BACKGROUND/AIM The cell line C6 is a continuous cell line of rat glioma and, as a transplantable line, is frequently used for induction into in vivo model of primary brain tumor. It is believed that, pursuant to its histological traits and biological behavior, this experimental tumor corresponds to human anaplastic astrocytoma of grade II/III, which is characterized by proliferative and invasive potency, and marked cell differentiation. The aim of this study was to determine macroscopic analysis of rat brain with implanted tumor during tumorigenesis, histological features of tumor cells of induced brain tumor and markers of proliferation (proliferation cell nuclear antigen - PCNA, cytokeratin - CK 19) and differentiation (glial fibrillary acidic protein -GFAP) in rat brain with implanted tumor. METHODS To determine histological structure of the brain with implanted C6 cells, we used brain sections stained for hematoxylin-eosin or kresyl violet, whereas other sections were immunohistochemically stained for GFAP, CK 19 and PCNA. RESULTS A statistically significant difference in weights of the left and right brain hemispheres with implanted tumors during tumorigenesis in as soon as 7 days from the day of inducing tumors was revealed. The tumor was of cellular type, with distinct pleomorphism of cells and frequent hyperchromasia of the nucleus. Immunohistochemical staining for PCNA revealed a significant number of positive cells on the days 7, 14 and 21 day following the implantation of C6 cells. CK 19 positive cells were present in both brain hemispheres, and numerous GFAP positive astrocytes were found around the puncture lesion. CONCLUSIONS Within the experimental conditions of the present research, C6 glioma did not demonstrate any relevant deviations concerning development, clinical symptomatology and macroscopic anatomy relative to those already described in the literature.

Objective: The suitability of the rabbit as an animal model for the primary screening and selection of the pilot scale batches during the early stages of the formulation development was studied. Materials and methods: Three modified-release formulations of aminophylline consisted of Carbopol® 971P/HPMC K4M (F-I), and HPMC K100M (F-II) or HPMC K4M (F-III) were used. Commercial products were Aminofilin retard 350 mg tablets, Srbolek, Serbia (R-I) and Phyllocontin® 350, tablets Purdue Frederic, Canada (R-II). Results: Calculated release rate constants and the ƒ2 values between R-I/F-I (84.1) and R-II/F-III (83.4) indicated similar in vitro release while the coefficient n showed presence of different mechanisms of release from Anomalous transport, Fickian diffusion to Case-II transport. Higher Tmax, was found in the rabbits, dosed with F-II (12.00 h), F-III (10.50 h), and R-II (15.00 h) formulation. The highest Cmax (9.22 mg/L) was obtained with F-II, similar lower values was seen for F-I and F-III, while commercial products showed the lowest values R-I (5.58 mg/L) and R-II (4.18 mg/L). Higher AUC values were detected for all three formulations (from 115.90 to 204.06 mgh/L) in relation to commercial products (105.33 and 113.25 mgh/L). Discussion and conclusion: The results demonstrated a good correlation of Level A (r2 = 0.97) for the two formulations (F-I, F-III) and commercial product (R-I) indicates that there is a reasonable assumption that the rabbit might be use as a model for the preliminary comparison of scale up formulations in the early stages of the product development.