The effect of the triazene compound CT913 on ovarian cancer cells in vitro and its synergistic interaction with the PARP-inhibitor olaparib.

OBJECTIVES: Extending the therapeutic spectrum of PARP-inhibitors (PARPi) beyond BRCA1-deficiency and/or overcoming PARPi-resistance is of high clinical interest. This is particularly true for the identification of innovative therapeutic strategies for ovarian cancer, given the recent advances in the use of PARPi in clinical practice. In this regard, the combination of PARPi with chemotherapy is a possible strategy for defining new therapeutic standards. In this study, we analyzed the therapeutic effect of novel triazene derivatives, including the drug CT913 and its metabolite CT913-M1 on ovarian cancer cells and describe their interaction with the PARPi olaparib. METHODS: In vitro assays for drug characterization including RNA-Seq were applied in a selected panel of ovarian cancer cell lines. RESULTS: CT913 treatment conferred a dose-dependent reduction of cell viability in a set of platinum-sensitive and platinum-resistant ovarian cancer cell lines with an IC50 in the higher micromolar range (553-1083 µM), whereas its metabolite CT913-M1 was up to 69-fold more potent, especially among long-term treatment (IC50 range: 8-138 µM). Neither of the drugs sensitized for cisplatin. CT913 conferred synthetic lethality in BRCA1-deficient ovarian cancer cells, indicating that its effect is augmented by a deficiency in homologous recombination repair (HR). Furthermore, CT913 showed a synergistic interaction with olaparib, independently of BRCA1 mutational status. CT913 strongly induced CDKN1A transcription, suggesting cell cycle arrest as an early response to this drug. It moreover downregulated a variety of transcripts involved in DNA-repair pathways. CONCLUSIONS: This is the first study, suggesting the novel triazene drug CT913 as enhancer drug for extending the therapeutic spectrum of PARPi.

Drug-induced xenogenization of tumors: A possible role in the immune control of malignant cell growth in the brain?

In recent years, immune checkpoint inhibitors (ICpI) have provided the ground to bring tumor immunity back to life thanks to their capacity to afford a real clinical benefit in terms of patient's survival. Essential to ICpI success is the presence of tumor-associated neoantigens generated by non-synonymous mutations, since a direct relationship between mutation load of malignant cells and susceptibility to ICpI has been confidently established. However, it has been also suggested that high intratumor heterogeneity (ITH) associated with subclonal neoantigens could not elicit adequate immune responses. Several years ago we discovered that in vivo treatment of leukemic mice with triazene compounds (TZC) produces a marked increase of leukemia cell immunogenicity [a phenomenon termed Drug-Induced Xenogenization (DIX)] through point mutations able to generate strong tumor neoantigens (Drug-Induced Neoantigens, DIN). Immunogenic mutations are produced by TZC-dependent methylation of O6-guanine of DNA, that is suppressed by the DNA repair protein methyl-guaninemethyltransferase (MGMT). This minireview illustrates preclinical investigations conducted in animal models where DIN-positive murine leukemia cells were inoculated intracerebrally into histocompatible mice. The analysis of the literature indicates that the growth of xenogenized malignant cells is controlled by anti-DIN graft responses and by intra-cerebral or intravenous adoptive transfer of anti-DIN cytotoxic T lymphocytes. This survey reminds also that PARP inhibitors increase substantially the antitumor activity of TZC and can be administered with the intent of suppressing more efficiently tumor load and possibly reducing ITH through downsizing the polyclonality of xenogenized tumor cell population. Finally, the present report illustrates a hypothetical clinical protocol that could be considered as an example of future development of DIXbased tumor immuno-chemotherapy in brain malignancies. The protocol involves oral or intravenous administration of TZC along with loco-regional (i.e. intracerebral "wafer") treatment with agents able to increase tumor cell sensitivity to the cytotoxic and xenogenizing effects of TZC (i.e. MGMT and PARP inhibitors) without enhancing the systemic toxicity of these DNA methylating compounds.

Triacsin C reduces lipid droplet formation and induces mitochondrial biogenesis in primary rat hepatocytes.

Intracellular long-chain acyl-CoA synthetases (ACSL) activate fatty acids to produce acyl-CoA, which undergoes ß-oxidation and participates in the synthesis of esterified lipids such as triacylglycerol (TAG). Imbalances in these metabolic routes are closely associated with the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Triacsin C is one of the few compounds that inhibit TAG accumulation into lipid droplets (LD) by suppressing ACSL activity. Here we report that treatment of primary rat hepatocytes with triacsin C at concentrations lower than the IC50 (4.1 µM) for LD formation: (i) diminished LD number in a concentration-dependent manner; (ii) increased mitochondrial amount; (iii) markedly improved mitochondrial metabolism by enhancing the ß-oxidation efficiency, electron transport chain capacity, and degree of coupling - treatment of isolated rat liver mitochondria with the same triacsin C concentrations did not affect the last two parameters; (iv) decreased the GSH/GSSG ratio and elevated the protein carbonyl level, which suggested an increased reactive oxygen species production, as observed in isolated mitochondria. The hepatocyte mitochondrial improvements were not related to either the transcriptional levels of PGC-1α or the content of mTOR and phosphorylated AMPK. Triacsin C at 10 µM induced hepatocyte death by necrosis and/or apoptosis through mechanisms associated with mitochondrial permeability transition pore opening, as demonstrated by experiments using isolated mitochondria. Therefore, triacsin C at sub-IC50 concentrations modulates the lipid imbalance by shifting hepatocytes to a more oxidative state and enhancing the fatty acid consumption, which can in turn accelerate lipid oxidation and reverse NAFLD in long-term therapies.

Exogenous nitric oxide enhances the prophylactic effect of aminoguanidine, a preferred iNOS inhibitor, on bleomycin-induced fibrosis in the lung: Implications for the direct roles of the NO molecule in vivo.

OBJECTIVE: Inducible nitric oxide synthase (iNOS) aggravates and endothelial nitric oxide synthase (eNOS) ameliorates fibrosis in the lung. Our previous study demonstrated that aminoguanidine (AG), a preferred iNOS inhibitor, prevents bleomycin-induced injury and fibrosis in the lung. The diethylenetriamine nitric oxide adduct (DETA/NO) is a slow-release NO donor. Here, to clarify the exact role of the nitric oxide (NO) molecule in the pathogenesis of pulmonary fibrosis in vivo, we observed the effects of inhalation of aerosolized DETA/NO on fibrosis in the lungs of bleomycin-exposed rats with AG treatment, including the effects on the myofibroblast number, collagen deposition, peroxynitrite anion (ONOO-) formation, and injury in the lung. DESIGN AND METHODS: Rats received a single intratracheal instillation of bleomycin or normal saline (NS) on day 0, followed by a daily intraperitoneal injection of AG or NS from day 1 to day 13. Each group was additionally given a daily inhalation of DETA/NO or placebo from day 1 to day 13. On day 14, half of the rats in each group was euthanized, and plasma nitrite and nitrate (NOx), myofibroblasts, type I collagen, ONOO- and injury in the lung were estimated by the Griess reaction, western blotting, immunohistochemical staining, sirius red staining, and hematoxylin and eosin (HE) staining, respectively. On day 28, the other half of the rats in each group was euthanized, and the total collagen of the lung was evaluated by hydroxyproline assay. RESULTS: ① At the day 14 time point, AG reduced the plasma NOx level in bleomycin rats, while this drug had no significant effect on sham rats. Inhalation of aerosolized DETA/NO increased the plasma NOx level of bleomycin + AG rats, sham rats and sham + AG rats. However, due to large areas of airspace obliteration in the lungs of bleomycin rats, DETA/NO inhalation had no significant effect on the plasma NOx level in these rats. ② At the day 14 time point, AG reduced ONOO- formation (marked by nitrotyrosine, NT), injury, myofibroblast number, and type I collagen deposition in the lungs of bleomycin rats, while this drug had no significant impact on the above parameters in the lungs of sham rats. Interestingly, DETA/NO inhalation enhanced the preventive effects afforded by AG on myofibroblast number and type I collagen deposition, but had no significant impact on ONOO- and injury in lung. ③ At the day 28 time point, because rats were not exposed to DETA/NO after day 13, there was no significant difference of the plasma NOx level in sham rats, sham + AG rats, bleomycin rats, and bleomycin + AG rats between DETA/NO inhalation and placebo inhalation. Interestingly, rats administered both DETA/NO and AG still showed a reduction in total collagen of the entire lung compared to rats administered AG alone at this time point. CONCLUSIONS: Exogenous NO enhances the prophylactic effect afforded by AG on the myofibroblast number and collagen deposition in the lungs of bleomycin-treated rats in vivo. These results suggest that NO has a direct antifibrotic effect in lungs, except for the formation of ONOO- in the development of pulmonary fibrosis in vivo.

Tumor immunotherapy: drug-induced neoantigens (xenogenization) and immune checkpoint inhibitors.

More than 40 years ago, we discovered that novel transplantation antigens can be induced in vivo or in vitro by treating murine leukemia with dacarbazine. Years later, this phenomenon that we called "Chemical Xenogenization" (CX) and more recently, "Drug-Induced Xenogenization" (DIX), was reproduced by Thierry Boon with a mutagenic/carcinogenic compound (i.e. N-methyl-N'-nitro-N-nitrosoguanidine). In both cases, the molecular bases of DIX rely on mutagenesis induced by methyl adducts to oxygen-6 of DNA guanine. In the present review we illustrate the main DIX-related immune-pharmacodynamic properties of triazene compounds of clinical use (i.e. dacarbazine and temozolomide).In recent years, tumor immunotherapy has come back to the stage with the discovery of immune checkpoint inhibitors (ICpI) that show an extraordinary immune-enhancing activity. Here we illustrate the salient biochemical features of some of the most interesting ICpI and the up-to-day status of their clinical use. Moreover, we illustrate the literature showing the direct relationship between somatic mutation burden and susceptibility of cancer cells to host's immune responses.When DIX was discovered, we were not able to satisfactorily exploit the possible presence of triazene-induced neoantigens in malignant cells since no device was available to adequately enhance host's immune responses in clinical settings. Today, ICpI show unprecedented efficacy in terms of survival times, especially when elevated mutation load is associated with cancer cells. Therefore, in the future, mutation-dependent neoantigens obtained by appropriate pharmacological intervention appear to disclose a novel approach for enhancing the therapeutic efficacy of ICpI in cancer patients.

Synthesis, characterization and biological activity of a gold(I) triazenide complex against chronic myeloid leukemia cells and biofilm producing microorganisms

ABSTRACT The enhancement of anti-leukemia therapy and the treatment of infections caused by multidrug-resistant pathogens are major challenges in healthcare. Although a large arsenal of drugs is available, many of these become ineffective, and as a result, the discovery of new active substances occurs. Notably, triazenes (TZCs) have been consolidated as a promising class of compounds, characterized by significant biological activity, especially antiproliferative and antimicrobial properties. The aim of this study is the synthesis and characterization of a new triazenide complex of gold (I), as well as the in vitro assessment of its antiproliferative activity against the K562 cell line (Chronic Myeloid Leukemia), and antibacterial activity against bacterial isolates of biofilm-producing coagulase-negative staphylococci. The combination of TZC with gold metal tends to have a synergistic effect against all biofilm-producing isolates, with Minimum Inhibitory Concentration values (MIC) between 32 and 64 µg mL-1. It has also shown activity against K562 cell line, getting an IC50=4.96 µM. Imatinib mesylate (Glivec) was used as reference, with IC50=3.86 µM. To the best of our knowledge, this study represents the first report of the activity of a TZC complexed with gold ion in the oxidation state (I) against microorganisms that produce biofilm and K562 cells.

Discovery and Clinical Evaluation of MK-8150, A Novel Nitric Oxide Donor With a Unique Mechanism of Nitric Oxide Release.

BACKGROUND: Nitric oxide donors are widely used to treat cardiovascular disease, but their major limitation is the development of tolerance, a multifactorial process to which the in vivo release of nitric oxide is thought to contribute. Here we describe the preclinical and clinical results of a translational drug development effort to create a next-generation nitric oxide donor with improved pharmacokinetic properties and a unique mechanism of nitric oxide release through CYP3A4 metabolism that was designed to circumvent the development of tolerance. METHODS AND RESULTS: Single- and multiple-dose studies in telemetered dogs showed that MK-8150 induced robust blood-pressure lowering that was sustained over 14 days. The molecule was safe and well tolerated in humans, and single doses reduced systolic blood pressure by 5 to 20 mm Hg in hypertensive patients. Multiple-dose studies in hypertensive patients showed that the blood-pressure-lowering effect diminished after 10 days, and 28-day studies showed that the hemodynamic effects were completely lost by day 28, even when the dose of MK-8150 was increased during the dosing period. CONCLUSIONS: The novel nitric oxide donor MK-8150 induced significant blood-pressure lowering in dogs and humans for up to 14 days. However, despite a unique mechanism of nitric oxide release mediated by CYP3A4 metabolism, tolerance developed over 28 days, suggesting that tolerance to nitric oxide donors is multifactorial and cannot be overcome solely through altered in vivo release of nitric oxide. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifiers: NCT01590810 and NCT01656408.

The Role of Nitric Oxide in the Antidepressant Actions of 5-Aminoimidazole-4-Carboxamide-1-ß-D-Ribofuranoside in Insulin-Resistant Mice.

OBJECTIVE: Depression and Type 2 diabetes mellitus are interrelated conditions, but the underlying neurobiology is insufficiently understood. The current study compared the effects of a pharmacological manipulation with 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR) that targets neurobiological processes by adenosine 5'-monophosphate-activated protein kinase activation versus exercise on depression-like behavior and nitric oxide (NO)-related measures. METHODS: A mouse model of a depression-like and insulin-resistant state, induced by the co-treatment of high-fat diet and corticosterone administration, was used to examine the antidepressant action of AICAR and exercise. RESULTS: Data showed that AICAR was a putative antidepressant in the depression-like and insulin-resistant mice (total ambulatory distance in the open-field test was 5120.69 ± 167.47 cm, mobility duration in the forced swim test was 17.61 ± 1.54 seconds, latency to feed in the novelty suppressed feeding test was 255.67 ± 37.80 seconds; all p values < .05). Furthermore, the antidepressant actions of AICAR required endothelial nitric oxide synthase activity with increased NO production in the prefrontal cortex, whereas corticosterone-induced expression of neuronal nitric oxide synthase and NO production may increase the risk of depression. In contrast to the traditional antidepressants such as ketamine and imipramine, AICAR interfered with the effects of insulin in skeletal muscle in the context of high-fat diet, consistent with the potential antidepressant effects of AICAR. Exercise also resulted in activation of adenosine 5'-monophosphate-activated protein kinase, nitric oxide synthase, and NO production (all p values < .01), which in turn may be implicated in the antidepressant effects of exercise. CONCLUSIONS: These findings suggest that NO is an essential signal mediating the antidepressant actions of AICAR. Ultimately, the concurrent effects of AICAR on brain insulin action and mitochondrial function suggest a potential of neural insulin resistance, which may contribute to our understanding of the comorbidity of depression and Type 2 diabetes.

Hepatoselective Nitric Oxide (NO) Donors, V-PYRRO/NO and V-PROLI/NO, in Nonalcoholic Fatty Liver Disease: A Comparison of Antisteatotic Effects with the Biotransformation and Pharmacokinetics.

V-PYRRO/NO [O(2)-vinyl-1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate] and V-PROLI/NO (O2-vinyl-[2-(carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate), two structurally similar diazeniumdiolate derivatives, were designed as liver-selective prodrugs that are metabolized by cytochrome P450 isoenzymes, with subsequent release of nitric oxide (NO). Yet, their efficacy in the treatment of nonalcoholic fatty liver disease (NAFLD) and their comparative pharmacokinetic and metabolic profiles have not been characterized. The aim of the present work was to compare the effects of V-PYRRO/NO and V-PROLI/NO on liver steatosis, glucose tolerance, and liver fatty acid composition in C57BL/6J mice fed a high-fat diet, as well as to comprehensively characterize the ADME (absorption, distribution, metabolism and excretion) profiles of both NO donors. Despite their similar structure, V-PYRRO/NO and V-PROLI/NO showed differences in pharmacological efficacy in the murine model of NAFLD. V-PYRRO/NO, but not V-PROLI/NO, attenuated liver steatosis, improved glucose tolerance, and favorably modified fatty acid composition in the liver. Both compounds were characterized by rapid absorption following i.p. administration, rapid elimination from the body, and incomplete bioavailability. However, V-PYRRO/NO was eliminated mainly by the liver, whereas V-PROLI/NO was excreted mostly in unchanged form by the kidney. V-PYRRO/NO was metabolized by CYP2E1, CYP2C9, CYP1A2, and CYP3A4, whereas V-PROLI/NO was metabolized mainly by CYP1A2. Importantly, V-PYRRO/NO was a better NO releaser in vivo and in the isolated, perfused liver than V-PROLI/NO, an effect compatible with the superior antisteatotic activity of V-PYRRO/NO. In conclusion, V-PYRRO/NO displayed a pronounced antisteatotic effect associated with liver-targeted NO release, whereas V-PROLI/NO showed low effectiveness, was not taken up by the liver, and was eliminated mostly in unchanged form by the kidney.

Triazene compounds in the treatment of acute myeloid leukemia: a short review and a case report.

Acute myeloid leukemia (AML) is a highly lethal disease, especially in old patients. Chemoresistance and the absence of host immune responses against autochthonous malignancy play a major role in the poor prognosis of AML. The triazene compounds Dacarbazine and Temozolomide are monofunctional alkylators that donate methyl groups to many sites in DNA, including the O(6)-position of guanine producing O(6)-methylguanine (O(6)-MeG). If not repaired, O(6)-MeG frequently mispairs with thymine during DNA duplication. O(6)-MeG:T mismatches can be recognized by the mismatch repair (MMR) system which activates a cascade of molecular events leading to cell cycle arrest and cell death. If MMR is defective, cells continue to divide and GC → AT transition mutations occur. In preclinical models, such mutations can lead to the appearance of abnormal proteins containing non-self peptides ("chemical xenogenization" CX) that can be recognized by host cell-mediated immunity. Repair of O(6)-MeG is achieved by the DNA repair protein, O(6)-methylguanine-DNA methyltransferase (MGMT), which removes the methyl adduct in an autoinactivating stoichiometric reaction. High MGMT levels attenuate the pharmacodynamic effects of triazenes. In the last few years, triazenes, alone or with MGMT inhibitors, have been tested in AML. In view of their potential activity as CX inducers, triazenes could offer the additional advantage of host anti-leukemia immune responses. The present paper describes several studies of leukemia treatment with triazenes and a case of acute refractory leukemia with massive skin infiltration by malignant cells. Treatment with Temozolomide and Lomeguatrib, a potent MGMT inhibitor, produced a huge, although transient, blastolysis and complete disappearance of all skin lesions.

[Five-year visual outcomes of typical age-related macular degeneration and/or polypoidal choroidal vasculopathy patients who received photodynamic therapy (PDT) as initial treatment in comparison with patients prior to the PDT era].

PURPOSE: To evaluate five-year visual outcomes of typical age-related macular degeneration (AMD) and polypoidal choroidal vasculopathy (PCV) in patients who received photodynamic therapy (PDT) as initial treatment compared with the outcomes of patients prior to the PDT era. SUBJECTS AND METHODS: Twenty-three eyes observed for 5 years before PDT was available (group A: typical AMD/PCV 16 eyes/7 eyes) and 61 eyes which had been observed for 5 years after PDT with additional treatment as needed (group B: typical AMD/PCV 25 eyes/36 eyes). The visual changes in these groups were retrospectively compared. RESULTS: In group A of typical AMD patients, the mean visual acuity (VA, logMAR) was significantly worse at the 3-year visit and later. In group B of typical AMD patients, the VA was stabilized after 2 years and no significant mean VA deterioration was observed for 5 years. More patients in group B retained a logMAR of less than 1.0 (43% vs. 25%) than in group A. Those patients in group B with PCV, maintained the VA for one year, but it gradually worsened thereafter. CONCLUSION: The PDT shortened the duration of VA deterioration in typical AMD patients from 5 to 2 years with no significant VA decrease for 5 years. The positive effect of PDT on PCV eyes was temporary.

Synthesis of triazenoazaindoles: a new class of triazenes with antitumor activity.

Despite improvements in the treatment and prevention of cancer, the number of new diagnoses continues to rise; this has fuelled substantial interest in the development of new and effective chemotherapeutic agents. Compounds of the triazene class, such as dacarbazine, have been used in the clinical management of many cancer types including brain, leukemia, and melanoma. A new compound class bearing a triazenoazaindole scaffold was synthesized with the aim of identifying new antiproliferative agents. Compounds 5a-g and 6a-c were screened against a panel of human tumor cell lines, and two of them, 5e and 5f, showed cytotoxicity (GI(50) range: 2.2-8.2 µM) in all cell lines. These two compounds even maintained their cytotoxicity in some multidrug-resistant cell lines. Flow cytometry analysis demonstrated their ability to induce cell death by apoptosis with involvement of lysosomes.

Molecular analysis of the in vivo metabolism and biodistribution of metabolically and non-metabolically activated combi-molecules of the triazene class.

Combi-molecules are novel agents designed to be hydrolyzed into two bioactive species: an epidermal growth factor receptor (EGFR) tyrosine kinase (TK) inhibitor + a DNA alkylating agent. With the purpose of enhancing the tumour concentration of the bioactive species, we synthesized and compared the activities of RB107, a quinazolinotriazene designed to generate the bioactive BJ2000 upon hydrolysis, ZRDM and RB107ZR that require metabolic activation to generate BJ2000. The results showed that RB107 released the highest level of BJ2000 and its degradation product FD105 in vivo and high levels of the DNA alkylating methyl diazonium ion in the brain, kidney, liver and the DU145 tumours as confirmed by (14)C-labeling. The results in toto suggest that RB107 was stable enough to deliver the bioactive species to the tumour site and for optimal tumour distribution of the bioactive species, combi-molecules of the triazene class must be designed to be primarily degraded by hydrolytic cleavage and not by metabolic activation.

Endothelial nitric oxide synthase plays an obligatory role in the late phase of ischemic preconditioning by activating the protein kinase C epsilon p44/42 mitogen-activated protein kinase pSer-signal transducers and activators of transcription1/3 pathway.

BACKGROUND: The role of endothelial nitric oxide synthase (eNOS) in ischemic preconditioning (PC) and cardioprotection is poorly understood. We addressed this issue using a genetic, rather than pharmacological, approach. METHODS AND RESULTS: In the nonpreconditioned state, eNOS-/- mice exhibited infarct sizes similar to those of wild-type mice. A sequence of six 4-minute coronary occlusion/4-minute reperfusion cycles (ischemic PC) induced late PC in wild-type mice; genetic deletion of eNOS abrogated the cardioprotection induced by late PC. In wild-type mice, ischemic PC induced membranous translocation of protein kinase C (PKC) epsilon and an increase in pSer-MEK-1/2 and pTyr-p44/42 mitogen-activated protein kinase, nuclear pSer-signal transducers and activators of transcription (STAT)1 and pSer-STAT3, and nuclear STAT1/3 DNA binding activity, followed by upregulation of cyclooxygenase-2 protein and activity 24 hours later. All of these changes were abrogated in eNOS-/- mice. The NO donor diethylenetriamine/NO recapitulated the effects of ischemic PC. CONCLUSIONS: In contrast to previous reports, we found that basal eNOS activity does not modulate infarct size in the nonpreconditioned state. However, eNOS is obligatorily required for the development of the cardioprotective effects of late PC and acts as the trigger of this process by activating the PKC epsilon-MEK-1/2-p44/42 mitogen-activated protein kinase pathway, leading to Ser-727 phosphorylation of STAT1 and STAT3 and consequent upregulation of STAT-dependent genes such as cyclooxygenase-2. The effects of eNOS-derived NO are reproduced by exogenous NO (NO donors), implying that nitrates can upregulate cardiac cyclooxygenase-2.

Influence of hydrocortisone, prednisolone, and NO association on the evolution of acute pancreatitis.

Leukocyte activation, inflammatory up-regulation, and microcirculatory disruption associated with ischemia-reperfusion injury are hallmarks in the pathogenesis of acute pancreatitis (AP). NO donors ensure microvascular integrity, while glucocorticoids act as anti-inflammatory and immune modulator drugs. AP was induced by the biliopancreatic duct outlet exclusion-closed duodenal loops (BPDOE-CDLs) model. Treatment with hydrocortisone (6 mg/kg) or prednisolone (0.5 mg/kg) alone or together with DETA-NO (0.5 mg/kg) was done (a)1 hr pre or (b)1 hr post, or (c) 1 hr pre and 4 hr post ,or (d) 4 hr post triggering AP. NOS inhibition by L-NAME (15 mg/kg) and glucocorticoid receptor blockage by mifepristone (3 mg/kg) was considered. AP severity was assessed by biochemical and histopathological analyses. Treatment with glucocorticoids together with DETA-NO 1 hr pre and 4 hr post BPDOE-CDLs reduced serum amylase, lipase, C-reactive protein, IL-6, IL-10, hsp72, and 8-isoprostane as well as pancreatic and lung myeloperoxidase. Acinar and fat necrosis, hemorrhage, and neutrophil infiltrate were also decreased. Hydrocortisone together with DETA-NO rendered the best results. We conclude that AP severity was significantly diminished by glucocorticoids associated with DETA-NO, with the optimal dose and time point of administration being crucial to provide adequate protection against AP.

p53-defective tumors with a functional apoptosome-mediated pathway: a new therapeutic target.

BACKGROUND: Although cancer cells appear to maintain the machinery for intrinsic apoptosis, defects in the pathway develop during malignant transformation, preventing apoptosis from occurring. How to specifically induce apoptosis in cancer cells remains unclear. METHODS: We determined the apoptosome activity and p53 status of normal human cells and of lung, colon, stomach, brain, and breast cancer cells by measuring cytochrome c-dependent caspase activation and by DNA sequencing, respectively, and we used COMPARE analysis to identify apoptosome-specific agonists. We compared cell death, cytochrome c release, and caspase activation in NCI-H23 (lung cancer), HCT-15 (colon cancer), and SF268 (brain cancer) cells treated with Triacsin c, an inhibitor of acyl-CoA synthetase (ACS), or with vehicle. The cells were mock, transiently, or stably transfected with genes for Triacsin c-resistant ACSL5, dominant negative caspase-9, or apoptotic protease activating factor-1 knockdown. We measured ACS activity and levels of cardiolipin, a mitochondrial phospholipid, in mock and ACSL5-transduced SF268 cells. Nude mice carrying NCI-H23 xenograft tumors (n = 10) were treated with Triacsin c or vehicle, and xenograft tumor growth was assessed. Groups were compared using two-sided Student t tests. RESULTS: Of 21 p53-defective tumor cell lines analyzed, 17 had higher apoptosome activity than did normal cells. Triacsin c selectively induced apoptosome-mediated death in tumor cells (caspase activity of Triacsin c-treated versus untreated SF268 cells; means = 1020% and 100%, respectively; difference = 920%, 95% CI = 900% to 940%; P<.001). Expression of ACSL5 suppressed Triacsin c-induced cytochrome c release and subsequent cell death (cell survival of Triacsin c-treated mock- versus ACSL5-transduced SF268 cells; means = 40% and 83%, respectively; difference = 43%, 95% CI = 39% to 47%; P<.001). ACS was also essential to the maintenance of cardiolipin levels. Finally, Triacsin c suppressed growth of xenograft tumors (relative tumor volume on day 21 of Triacsin c-treated versus untreated mice; means = 4.6 and 9.6, respectively; difference = 5.0, 95% CI = 2.1 to 7.9; P = .006). CONCLUSIONS: Many p53-defective tumors retain activity of the apoptosome, which is therefore a potential target for cancer chemotherapy. Inhibition of ACS may be a novel strategy to induce the death of p53-defective tumor cells.

The selective pulmonary vasodilatory effect of inhaled DETA/NO, a novel nitric oxide donor, in ARDS-a pilot human trial.

OBJECTIVES: To examine the effects of inhaled NONOates in patients with acute respiratory distress syndrome (ARDS). DESIGN: Case-series, phase I clinical trial. SETTING: A multidisciplinary intensive care unit in a tertiary teaching hospital. PATIENTS: Five consecutive patients with ARDS (men; age range, 47-76 years). MEASUREMENTS: DETA/NO (150 micromol) was aerosolized into the lungs of patients on mechanical ventilation via the endotracheal tube over 20 minutes. Hemodynamic parameters were measured and blood samples were taken before, during, and after inhalation. RESULTS: Compared to baseline values, pulmonary vascular resistance decreased until the end of the study period (180 minutes) while intrapulmonary shunting decreased significantly up to 45 min after DETA/NO aerosol administration. Inhaled DETA/NO had no effect on the systemic circulation (systemic blood pressure or cardiac output). CONCLUSIONS: Inhaled DETA/NO is a selective pulmonary vasodilator in patients with ARDS. However, a larger number of patients is required to confirm the findings of this pilot study.

Effects of a nitric oxide donor on and correlation of changes in cyclic nucleotide levels with experimental vasospasm.

OBJECTIVE: Vasospasm after subarachnoid hemorrhage (SAH) may result from hemoglobin-mediated removal of nitric oxide (NO) from the arterial wall. We tested the ability of the long-acting, water-soluble, NO donor (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-1,2-diolate (DETA/NO), delivered via continuous intracisternal infusion, to prevent vasospasm in a nonhuman primate model of SAH. METHODS: First, vasorelaxation in response to DETA/NO was characterized in vitro by using monkey basilar artery rings under isometric tension. Next, monkeys were randomized to undergo angiography, unilateral SAH, and no treatment (SAH only, n = 4) or treatment with DETA/NO (1 mmol/L, 12 ml/d, n = 4) or decomposed DETA/NO (at the same dose, n = 4). Vasospasm was assessed by angiography, which was performed on Day 0 and Day 7. Levels of cyclic adenosine monophosphate and cyclic guanosine monophosphate (cGMP) were measured in cerebral arteries on Day 7. RESULTS: DETA/NO produced significant relaxation of monkey arteries in vitro, which reached a maximum at concentrations of 10(-5) mol/L. In monkeys, angiography demonstrated significant vasospasm of the right intradural cerebral arteries in all three groups, with no significant difference in vasospasm among the groups (P > 0.05, analysis of variance). The ratios of cGMP or cyclic adenosine monophosphate levels in the right and left middle cerebral arteries were not different among the groups (P > 0.05, analysis of variance). There was no significant correlation between arterial cGMP contents and the severity of vasospasm. CONCLUSION: DETA/NO did not prevent vasospasm. There was no correlation between the severity of vasospasm and cyclic adenosine monophosphate and cGMP levels in the cerebral arteries. These results suggest that events downstream of cyclic nucleotides may be abnormal during vasospasm.

Spin labelled nitrosoureas and triazenes and their non-labelled clinically used analogues--a comparative study on their physicochemical properties and antimelanomic effects.

Physicochemical properties, such as half life time (tau0.5), alkylating and carbamoylating activity and in vivo antimelanomic effects against B16 melanoma of spin labeled (containing nitroxyl free radical moiety) amino acid nitrosoureas, synthesized in our laboratory, have been studied and compared to those of the antitumor drug N'-cyclohexyl-N-(2-chloroethyl)-N-nitrosourea (lomustine, CCNU). We have shown that the introduction of amino acid moieties and the replacement of cyclohexylamine with nitroxyl moiety leads to a faster decomposition, higher alkylating, lower carbamoylating activity, better antimelanomic activity and lower general toxicity, when compared to those of CCNU. It was also established that spin labeled triazenes, previously synthesized by us, were more stable in phosphate saline than their nonlabeled analogue, 5-(3,3-dimethyltriazene-1-yl)-imidazole-4-carboxamide (dacarbazine, DTIC). A higher cytotoxicity to B16 melanoma cells than to YAC-1 and lymphocytes was demonstrated for all spin labeled triazenes, in comparison with DTIC. An assumption has been made to explain the lower general toxicity of the spin labeled nitrosoureas compared to that of CCNU. Based on the results presented, we accept that a new trend for synthesis of more selective and less toxic nitrosourea and triazene derivatives as potential antimelanomic drugs might be developed.