Different patterns of H2S/NO activity and cross-talk in the control of the coronary vascular bed under normotensive or hypertensive conditions.

Hydrogen sulfide (H2S) and nitric oxide (NO) play pivotal roles in the cardiovascular system. Conflicting results have been reported about their cross-talk. This study investigated their interplays in coronary bed of normotensive (NTRs) and spontaneously hypertensive rats (SHRs). The effects of H2S- (NaHS) and NO-donors (sodium nitroprusside, SNP) on coronary flow (CF) were measured in Langendorff-perfused hearts of NTRs and SHRs, in the absence or in the presence of propargylglycine (PAG, inhibitor of H2S biosynthesis), L-NAME (inhibitor of NO biosynthesis), ODQ (inhibitor of guanylate cyclase), L-Cysteine (substrate for H2S biosynthesis) or L-Arginine (substrate for NO biosynthesis). In NTRs, NaHS and SNP increased CF; their effects were particularly evident in Angiotensin II (AngII)-contracted coronary arteries. The dilatory effects of NaHS were abolished by L-NAME and ODQ; conversely, PAG abolished the effects of SNP. In SHRs, high levels of myocardial ROS production were observed. NaHS and SNP did not reduce the oxidative stress, but produced clear increases of the basal CF. In contrast, in AngII-contracted coronary arteries of SHRs, significant hyporeactivity to NaHS and SNP was observed. In SHRs, the vasodilatory effects of NaHS were only modestly affected by L-NAME and ODQ; PAG poorly influenced the effects of SNP. Then, in NTRs, the vascular actions of H2S required NO and vice versa. By contrast, in SHRs, the H2S-induced actions scarcely depend on NO release; as well, the NO effects are largely H2S-independent. These results represent the first step for understanding pathophysiological mechanisms of NO/H2S interplays under both normotensive and hypertensive conditions.

Effect of acute and chronic vitamin D administration on systemic renin angiotensin system in essential hypertensives and controls.

AIM: To investigate the systemic renin-angiotensin system (RAS) in essential hypertensives (EH) and controls (C) after short- and long-term vitamin D receptor activation. DESIGN: Ten consecutive EH (under controlled low-salt diet) and 10 C underwent calcitriol administration (0.25 µg bid) for 1 week (Group A). Eighteen consecutive EH under angiotensin II receptor antagonist therapy received a single oral dose of 300,000 IU of cholecalciferol and were followed up for 8 weeks (Group B). METHODS: In basal conditions and at the end of the study (1 week in Group A and 8 weeks in Group B), plasma renin activity (PRA), plasma active renin, aldosterone, and angiotensin II were evaluated, as well as blood pressure, plasma 25-hydroxyvitamin D [25(OH)D], 1,25-dihydroxyvitamin D [1,25(OH)2D], and PTH. RESULTS: In Group A, plasma 25(OH)D levels in EH and C were below the normal range, although lower levels were found in the former. No association between basal plasma 25(OH)D or 1,25(OH)2D levels and blood pressure values or RAS components was observed either in the whole group or in the two subgroups. Calcitriol administration did not affect any RAS parameter either in EH or in C. In Group B, cholecalciferol significantly increased 25(OH)D and 1,25(OH)2D levels without interfering with the angiotensin II receptor antagonist-induced increase in RAS components. No correlation was found between plasma 25(OH)D or 1,25(OH)2D levels and blood pressure values or RAS parameters before and after cholecalciferol administration. CONCLUSIONS: The present data suggest that, in our experimental conditions, vitamin D receptor activation is unable to influence systemic RAS activity.

Polymethylmethacrylate strengthens antibody response in hemodialysis patients not responding to hepatitis B vaccine: preliminary data.

AIM: Most chronic uremic patients undergoing dialysis show a diminished antibody response to anti-hepatitis B vaccination (anti-HBv) and at this regard high sCD40 serum levels seem to inhibit the immunocompetent response. A simple and feasible technique for removing soluble sCD40 by adsorption is standard bicarbonate dialysis performed by polymethylmethacrylate (PMMA) dialyzers able to remove serum high molecular weight toxins. Aim of the study was to assess whether PMMA dialyzers could enhance the long-term response to anti-HBv in non-responder (NR) ESRD patients. METHODS: The study involved 5 ESRD patients (mean age 65 years) who had been on maintenance thrice-weekly dialysis for 42.4±15.8 months. They had been previously defined NR to anti-HBv for their low antibody titres (<10 IU/L) despite a previous full course of vaccination using protocols recommended for patients on dialysis. After a period of three months of treatment with PMMA dialyzers, keeping all the parameters of dialysis efficiency (the filter surface of 2.1 m(2), KT/V, dialysis time, QB) unchanged compared to previous treatments, all patients received a new booster dose of Fendrix vaccine (20 µg). Hepatitis B markers were checked at month 1, 3, 12 and 18 after administration of the booster dose and returning to the previous membranes used. RESULTS: Three of five patients showed a strong antibody response (>1000 IU/L) that lasted effectively over time even after 18 months despite discontinuation of PMMA. CONCLUSION: In conclusion our preliminary results confirm that PMMA membranes enhance immune response to anti-HBv in NR HD patients.

Wild ungulate slaughtering and meat inspection.

The different phases of production of farmed and hunted wild game fresh meat are described. The importance of reducing the stress resulting from handling procedures (capture, restraint, transport) before the slaughtering of animals is highlighted, due to its adverse effects on meat quality. The hygienic and animal welfare criteria to be adopted in the slaughtering of wild game are described. The importance of carcass inspection immediately after slaughtering is stated, so that meat can be destined for human consumption. Possible alterations occurring in fresh and refrigerated meat, that are capable of compromising its consumability, are presented.

Tyr1068-phosphorylated epidermal growth factor receptor (EGFR) predicts cancer stem cell targeting by erlotinib in preclinical models of wild-type EGFR lung cancer.

Tyrosine kinase inhibitors (TKIs) have shown strong activity against non-small-cell lung cancer (NSCLC) patients harboring activating epidermal growth factor receptor (EGFR) mutations. However, a fraction of EGFR wild-type (WT) patients may have an improvement in terms of response rate and progression-free survival when treated with erlotinib, suggesting that factors other than EGFR mutation may lead to TKI sensitivity. However, at present, no sufficiently robust clinical or biological parameters have been defined to identify WT-EGFR patients with greater chances of response. Therapeutics validation has necessarily to focus on lung cancer stem cells (LCSCs) as they are more difficult to eradicate and represent the tumor-maintaining cell population. Here, we investigated erlotinib response of lung CSCs with WT-EGFR and identified EGFR phosphorylation at tyrosine1068 (EGFRtyr1068) as a powerful biomarker associated with erlotinib sensitivity both in vitro and in preclinical CSC-generated xenografts. In contrast to the preferential cytotoxicity of chemotherapy against the more differentiated cells, in EGFRtyr1068 cells, erlotinib was even more active against the LCSCs compared with their differentiated counterpart, acquiring potential value as CSC-directed therapeutics in the context of WT-EGFR lung cancer. Although tumor growth was inhibited to a similar extent during erlotinib or chemotherapy administration to responsive tumors, erlotinib proved superior to chemotherapy in terms of higher tolerability and reduced tumor aggressiveness after treatment suspension, substantiating the possibility of preferential LCSC targeting, both in adenocarcinoma (ADC) and squamous cell carcinoma (SCC) tumors. We conclude that EGFRtyr1068 may represent a potential candidate biomarker predicting erlotinib response at CSC-level in EGFR-WT lung cancer patients. Finally, besides its invariable association with erlotinib sensitivity in EGFR-WT lung CSCs, EGFRtyr1068 was associated with EGFR-sensitizing mutations in cell lines and patient tumors, with relevant diagnostic, clinical and therapeutic implications.

Structural requirements for inhibitors of poly(ADP-ribose) polymerase.

The purpose of this study was to examine the structure/activity relationships of a series of substituted benzamides as poly(ADP-ribose) polymerase inhibitors. The experimental approach has involved the use of in vitro and in vivo assays in order to gather information either on the intrinsic activity of the benzamides or on the effect of various pharmacodynamic parameters on the activity in vivo. Although some discrepancies between the data obtained in vivo and in vitro were found in this study, results seem to indicate that most powerful inhibitors were characterized by acylation of the -NH2 function in the 3 position or by substitution in this same position with hydroxy or methoxy groups. The best inhibitors were not cytotoxic under these experimental conditions. Computed calculations of molecular electrostatic potential of these molecules were also performed and a good correlation was found between the similarity index and the experimental inhibitory activity.

Inhibition by nomegestrol acetate and other synthetic progestins on proliferation and progesterone receptor content of T47-D human breast cancer cells.

Progesterone receptors (PgR) of human breast cancer T47-D cells grown in an estrogenic environment (presence of phenol red, natural estrogens of foetal calf serum and insulin) were found to be present in considerable amounts (1-3 pmol/mg protein and 20 pmol/mg DNA), and to specifically bind progestins with a high affinity characterized by a Kd around 3 nM for ORG2058, and 4 nM for nomegestrol acetate (NOM; 17 alpha-acetoxy-6-methyl-19-nor-pregna-4,6-diene-3, 20-dione), when measured under equilibrium conditions. Both compounds formed an highly stable ligand-receptor complex with a dissociation constant (k-1) around 1 x 10(-5) s-1. At high pharmacological concentrations, NOM, ORG2058 and other synthetic progestins including promegestone (R5020), medroxy-progesterone acetate and norethindrone acetate (NOR), induced a dose-dependent inhibition of cell proliferation as measured by [3H]thymidine incorporation. Dexamethasone, which did not bind to PgR, did not reproduce this inhibitory effect. NOM, R5020 and NOR treatments of T47-D cells at concentrations around Kd resulted in an 80% decrease in PgR content. Our data on NOM as compared to other progestins are consistent with their antiproliferative effects on human breast cancer cells grown in estrogenic conditions.

Lack of estrogenic potential of progesterone- or 19-nor-progesterone-derived progestins as opposed to testosterone or 19-nor-testosterone derivatives on endometrial Ishikawa cells.

Estrogen receptors of human endometrial cancer Ishikawa cells were found to be present in moderate amounts (160-200 fmol/mg protein), and to specifically bind moxestrol (R2858) with a very high affinity characterized by a Kd around 60 pM, when measured under equilibrium conditions. The binding specificity respected a decreasing order as follows: estradiol (E2: 100%) > 4-hydroxy-tamoxifen (4OHTAM: 52.7%) > estriol (E3: 5.7%) > estrone (E1: 2.1%) > TAM (0.2%). The induction of alkaline phosphatase activity (APase) used as an estrogen-specific response, confirmed the intrinsic estrogenicity of progestins derived from 19-nor-testosterone (19NT): norethindrone (NOR), norethynodrel and levonorgestrel, at concentrations ranging from 10(-8) to 10(-6) M. The effect of NOR was partially blocked by the antiestrogen 4OHTAM, which was also partially agonistic in this model, but neither by the antiprogestin mifepristone (RU486) nor by the aromatase inhibitor aminoglutethimide. A simulatory effect was also detected at 10(-7) or 10(-6) M with ethindrone, the testosterone- (T) derived progestin homologous to NOR, and with both androgenic parent-compounds, i.e. T and 19NT themselves. In contrast, progesterone (P) derivatives like medroxyprogesterone acetate (MPA) and chlormadinone acetate (CMA) remained totally inactive, as well as 19-nor-progesterone (19NP) itself or its progestagenic derivatives: ORG 2058 and nomegestrol acetate (NOM). Structure-activity relationships deduced from these studies suggest that it is not the absence of the 19-methyl group which can account for the estrogenic potential of the so-called "19-norprogestins", but rather their steroid structure derived from T in a broad sense (including the 19NT derivatives), as opposed to the non-estrogenic therapeutic progestins derived from P like MPA or CMA, or from 19NP like NOM.

An overview of nomegestrol acetate selective receptor binding and lack of estrogenic action on hormone-dependent cancer cells.

The specific pharmacological profile of the 19-norprogestin nomegestrol acetate (NOMAC) is, at least in part, defined by its pattern of binding affinities to the different steroid hormone receptors. In the present study, its affinity to the progesterone receptor (PgR), the androgen receptor (AR) and the estrogen receptor (ER) was re-evaluated and compared to those obtained for progesterone (P) and several progestins. The characteristics of binding to the PgR in rat uterus were determined and Ki were found to be roughly similar with 22.8 and 34.3 nM for NOMAC and P, respectively. The binding characteristics of 3H-NOMAC were also determined and compared to that of 3H-ORG2058 with Kd of 5 and 0.6 nM, respectively for rat uterus and 4 and 3 nM, respectively for human T47-D cells. Structure-affinity and -activity relationships were studied on a variety of compounds related to NOMAC in order to assess its specificity as a progestin. The effects of NOMAC on the binding of androgen to the AR were investigated, using rat ventral prostate as target model. Contrary to what was observed for MPA, the RBA of NOMAC was found to decline with time, showing anti-androgenic rather than androgenic potential, a result that was confirmed in vivo. Regarding the ER, since none of the progestins were able to compete with estrogen for binding in rat uterus as well as in Ishikawa cells, the induction of alkaline phosphatase activity (APase) was used as an estrogen-specific response. It confirmed the intrinsic estrogenicity of progestins derived from 19-nor-testosterone (19NT), norethisterone acetate (NETA), levonorgestrel (LNG) or norgestimate (NGM) and others. In contrast, all P and 19-norP derivatives remained inactive. Finally, to complete this overview of NOMAC at the sex steroid receptor levels, the lack of estrogenic or estrogenic-like activity was checked out in different in vitro models. Data from this study have demonstrated that NOMAC is a progestin that has greater steroid receptor selectivity compared to MPA or some other synthetic progestins. It may provide a better pharmacological profile than those progestins currently in use in HRT and OC.

In vitro and in vivo models for the evaluation of new inhibitors of human steroid sulfatase, devoid of residual estrogenic activity.

The goal of our research project is to develop a new class of orally active drugs, estrone sulfatase inhibitors, for the treatment of estrogen-dependent (receptor positive) breast cancer. Several compounds were synthesized and their pharmacological potencies explored. Based on encouraging preliminary results, three of them, TX 1299, TX 1492 and TX 1506 were further studied in vitro as well as in vivo. They proved to be strong inhibitors of estrone sulfatase when measured on the whole human JEG-3 choriocarcinoma and MCF-7 breast cancer cells and their IC(50)s found to be in the range of known standard inhibitors. Their residual estrogenic activity was checked as negative in the test of induction of alkaline phosphatase (APase) activity in whole human endometrial adenocarcinoma Ishikawa cells. In addition, their effect on aromatase activity in JEG-3 cells was also examined, since the goal of inhibiting both sulfatase and aromatase activities appears very attractive. However, it has been unsuccessful so far. Then, in vivo potencies of TX 1299, the lead compound in our chemical series, were evaluated in comparison with 6,6,7-COUMATE, a non-steroidal standard, in two different rat models and by oral route. First, the absence of any residual estrogenic activity for these compounds was checked in the uterotrophic model in prepubescent female rats. Second, antiuterotrophic activity in adult ovariectomized rat supplemented with estrone sulfate (E(1)S), showed that both compounds were potent inhibitors, the power of TX 1299 relative to 6,6,7-COUMATE being around 80%. This assay was combined with uterine sulfatase level determination and confirmed the complete inhibition of this enzyme within the target organ. Preliminary studies indicated that other non-steroid compounds in the Théramex series were potent in vitro and in vivo inhibitors of estrone sulfatase in rats and further studies are in progress.

Is hypertension a mortality risk factor in dialysis?

We retrospectively studied 370 chronic uremic patients, 234 males and 136 females with a mean age of 53 +/- 15 years, to determine the number of mortalities due to hypertension. With hypertension defined as blood pressure values > 150/90, a total of 168 patients were found to be normotensive and 202 as hypertensive. Blood pressure was also considered in association with some prognostic variables such as the patient's age, time of dialysis, renal diagnosis, and dialytic treatment (hemodialysis or peritoneal dialysis). No significant difference in survival was found between normotensive and hypertensive patients. Patients with diabetic nephopathy had a significantly poorer survival experience with respect to other nephropathies, independently of blood pressure values after beginning dialysis treatment. The Cox proportional hazard analysis showed an increased risk of death from aging and peritoneal dialysis, while the chi 2 test showed the role of hypertension as a mortality risk factor only in patients less than 50 years old (18% of deaths among normotensives vs. 31% of deaths among hypertensives, P < 0.05). We conclude that hypertension does not seem to represent the primary risk factor for overall survival in dialysis therapy.

2-Bromomelatonin: synthesis and characterization of a potent melatonin agonist.

A practical synthesis of N-[2-(2-bromo-5-methoxy-1H-indol-3-yl)ethyl]- acetamide (2-bromomelatonin) was achieved by direct bromination of melatonin with N-bromosuccinimide (NBS) in anhydrous acetic acid at room temperature under nitrogen, followed by flash-chromatography. 1H-NMR and mass spectra showed the bromine to be incorporated at the C-2 position of the indole moiety. Tests performed in vitro with isolated melatonin receptors from rabbit parietal cortex demonstrated that the relative binding affinity of 2-bromomelatonin was about ten times higher than that of melatonin and close to that of 2-iodomelatonin. 2-Bromomelatonin behaved as a potent agonist in the physiological studies. It showed enhanced activity in inhibiting the spontaneous firing activity of cortical neurons and similarly to melatonin and 2-iodomelatonin potentiated significantly the inhibitory effect of GABA. 2-Bromomelatonin was also an extremely effective agonist in the tests performed in vivo in the Syrian hamster gonadal regression model.

Ethylene-oxide and steam-sterilised polysulfone membrane in dialysis patients with eosinophilia.

Eosinophilia and some acute dialysis side-effects, such as itching, flushing and bronchospasm, are often associated with the presence of ethylene oxide (ETO) as dialyzer sterilizing agent. This study evaluated the effects of two different polysulfone (PS) hollow-fiber dialysers sterilized with ETO and steam in 31 chronic dialysis patients with eosinophilia. Clinical symptoms, metabolic and biochemical parameters, complement (C3a and C5a) activation and production were evaluated in each patient dialysed for two months at a time with Cuprophan dialyser, ETO-PS dialyser and steam-PS dialyser. The steam-sterilizer agent does not alter the purifying capacity of the PS membrane which maintains its superiority over Cuprophan in terms of biocompatibility. Using steam-PS, intradialytic eosinophil kinetics seems to improve. In some patients with high serum levels of ETO-specific IgE these levels tend to diminish. Generic intradialytic symptoms do not differ between the two sterilization methods, although some hypersensitivity symptoms during the first dialysis hour are considerably lower in some patients when steam-sterilized PS is used.

Serum B2 microglobulin (B2M) in CAPD.

Twenty-six chronic uremic pts (8F and 18M, mean age 64 yrs) on CAPD for 15 months on the average, were studied as regards the levels of B2M measured by radioimmunoassay in fasting serum samples. Considering residual renal function, 2 pts groups were identified: 11 pts with a diuresis greater than or equal to 500 cc/day and 15 pts with a diuresis less than 500 cc/day. Serum B2M was significantly decreased in the first group (24.8 +/- 8.1 vs 34.7 +/- 7.1 mg/l; p less than 0.02). Considering the incidence of peritonitis episodes, 12 pts without and 14 pts with one or more peritonitis episodes were noted. The group with no peritonitis showed significantly decreased serum B2M values (25.1 +/- 7.6 vs 35.03 +/- 4.8 mg/l; p less than 0.005). No relationship was found as regards primary renal diseases, sex and age of pts. We conclude that residual renal function and peritonitis episodes modify serum B2M levels in CAPD. The long term follow up of pts could be of interest for better defining the role of these factors as regards the incidence of pathologic lesions associated with B2M deposition.

7-Substituted theophyllines. Part VI--N-[2-(4-chlorophenoxy)-1-phenyl-alkyl]-alkylamido derivatives.

The new amides are inhibitors of cholesterol biosynthesis in vitro as well as in vivo. These compounds are also inhibitors in vivo of triglyceride biosynthesis and of platelet aggregation. All tests showed activity (values expressed as percentage variation) much greater than clofibrate. Besides, all compounds have no effect on coagulation or diuresis and showed low acute toxicity.

Methyl N-(diphenylmethyl)-D,L-tryptophanates: synthesis and benzodiazepine receptor affinity.

Several Methyl N-(diphenylmethyl)-D,L-tryptophanates were synthetized and the affinity for the central benzodiazepine receptor was measured. Disappointingly, none of the tested compounds showed to be active, even at the high concentration examined.

N-benzhydryl-tryptamines and 1,1-diphenyl-tetrahydro-beta-carbolines: synthesis, X-ray crystal structure and benzodiazepine receptor affinity.

Several N-benzhydryl-tryptamines and 1,1-diphenyl-tetrahydro-beta-carbolines were synthetized and the requisites for their formation were established. Crystallographic and conformational analyses were carried out on selected compounds and the affinity for the central benzodiazepine receptors was measured.

Therapeutic targeting of Chk1 in NSCLC stem cells during chemotherapy.

Cancer stem cell (SC) chemoresistance may be responsible for the poor clinical outcome of non-small-cell lung cancer (NSCLC) patients. In order to identify the molecular events that contribute to NSCLC chemoresistance, we investigated the DNA damage response in SCs derived from NSCLC patients. We found that after exposure to chemotherapeutic drugs NSCLC-SCs undergo cell cycle arrest, thus allowing DNA damage repair and subsequent cell survival. Activation of the DNA damage checkpoint protein kinase (Chk) 1 was the earliest and most significant event detected in NSCLC-SCs treated with chemotherapy, independently of their p53 status. In contrast, a weak Chk1 activation was found in differentiated NSCLC cells, corresponding to an increased sensitivity to chemotherapeutic drugs as compared with their undifferentiated counterparts. The use of Chk1 inhibitors in combination with chemotherapy dramatically reduced NSCLC-SC survival in vitro by inducing premature cell cycle progression and mitotic catastrophe. Consistently, the co-administration of the Chk1 inhibitor AZD7762 and chemotherapy abrogated tumor growth in vivo, whereas chemotherapy alone was scarcely effective. Such increased efficacy in the combined use of Chk1 inhibitors and chemotherapy was associated with a significant reduction of NSCLC-SCs in mouse xenografts. Taken together, these observations support the clinical evaluation of Chk1 inhibitors in combination with chemotherapy for a more effective treatment of NSCLC.