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The corona virus pandemic has ignited a proliferation of research aimed at prevention of spread, early diagnosis and treatment. Coincidentally, in recent years the Yorkshire Laser Centre has been engaged in developing the methodology of applying PDT in chronic bronchiectasis. Our methodology is based on Methylene Blue (MB) mediated PDT used topically within the airway. The novelty of the method is the use of a nebulizer to deliver the photosensitizer. We suggest that our protocol and methodology could be modulated for use in respiratory infections of COVID -19., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Nitric oxide (NO) release in the right medial prefrontal cortex (RmPFC) produces anxiogenesis. In the bed nucleus of the stria terminalis (BNST), a region that receives neuronal projections from the mPFC, NO provokes anxiety, an effect that is blocked by local injections of corticotrophin-releasing factor type 1 receptor (CRF 1 ) or n-methyl-d-aspartate receptor (NMDAr) antagonist. Anxiety is also enhanced by social defeat stress, and chronic stress impairs and facilitates, respectively, PFC and BNST roles in modulating behavioral responses to aversive situations. This study investigated whether the (i) chronic social defeat stress (CSDS) increases NO signaling in the mPFC; and/or (ii) anxiogenic effects provoked by the intra-RmPFC injection of NOC-9 (an NO donor) or by CSDS are prevented by intra-BNST injections of AP-7 (0.05 nmol) or CP 376395 (3.0 nmol), respectively, NMDAr and CRF 1 antagonists, in male Swiss-Webster mice exposed to the elevated plus-maze (EPM). Results showed that (a) CSDS increased anxiety (i.e., reduced open-arm exploration) and repeatedly activated nNOS-containing neurons, as measured by ΔFosB (a stable nonspecific marker of neural activity) + nNOS double-labeling, in the right (but not left) mPFC, (b) NOC-9 in the RmPFC also increased anxiety, and (c) both CSDS and NOC-9 effects were reversed by injections of AP-7 or CP 376395 into the BNST. These results suggest that NMDA and CRF 1 receptors located in BNST play an important role in the modulation of anxiety provoked by NO in the RmPFC, as well as by chronic social defeat in mice., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: The homeostasis of lipid droplets (LDs) plays a crucial role in maintaining the physical metabolic processes in cells, and is regulated by many LD-associated proteins, including perilipin 5 (Plin5) in liver. As the putative sites of hepatitis C virus (HCV) virion assembly, LDs are vital to viral infection. In addition, the hepatic LD metabolism can be disturbed by non-structural HCV proteins, such as NS5A, but the details are still inexplicit., Methods: HCV NS5A was overexpressed in the livers and hepatocytes of wild-type and Plin5-null mice. BODIPY 493/503 and oil red O staining were used to detect the lipid content in mouse livers and hepatocytes. The levels of lipids, lipid peroxidation and inflammation biomarkers were further determined. Immunofluorescence assay and co-immunoprecipitation assay were performed to investigate the relationship of Plin5 and NS5A., Results: One week after adenovirus injection, livers expressing NS5A showed more inflammatory cell aggregation and more severe hepatic injuries in Plin5-null mice than in control mice, which was consistent with the increased serum levels of IL-2 and TNF-α (P < 0.05) observed in Plin5-null mice. Moreover, Plin5 deficiency in the liver and hepatocytes aggravated the elevation of MDA and 4-HNE levels induced by NS5A expression (P < 0.01). The triglyceride (TG) content was increased approximately 25% by NS5A expression in the wild-type liver and hepatocytes but was unchanged in the Plin5-null liver and hepatocytes. More importantly, Plin5 deficiency in the liver and hepatocytes exacerbated the elevation of non-esterified fatty acids (NEFAs) stimulated by NS5A expression (P < 0.05 and 0.01 respectively). Using triacsin C to block acyl-CoA biosynthesis, we found that Plin5 deficiency aggravated the NS5A-induced lipolysis of TG. In contrast, Plin5 overexpression in HepG2 cells ameliorated the NS5A-induced lipolysis and lipotoxic injuries. Immunofluorescent staining demonstrated that NS5A expression stimulated the targeting of Plin5 to the surface of the LDs in hepatocytes without altering the protein levels of Plin5. By co-IP, we found that the N-terminal domain (aa 32-128) of Plin5 was pivotal for its binding with NS5A., Conclusions: Our data highlight a protective role of Plin5 against hepatic lipotoxic injuries induced by HCV NS5A, which is helpful for understanding the steatosis and injuries in liver during HCV infection.

Vascular endothelial dysfunction and platelet activation play a key role in tumor metastasis, and therefore, both of these processes are considered important therapeutic targets in cancer. The aim of our studies was to analyze antimetastatic activity of combination therapy using nitric oxide donor DETA/NO and antiplatelet drug clopidogrel. Nitric oxide acts as a vasoprotective mediator, while clopidogrel inhibits ADP-mediated platelet aggregation. 4T1-luc2-tdTomato cell line transplanted intravenously (i.v.) and 4T1 cell line transplanted orthotopically were used as metastatic mammary gland cancer models. Moreover, antiaggregation action of compounds was tested ex vivo on the blood samples taken from breast cancer patients. We have shown that in selected dosage regimes, DETA/NO combined with clopidogrel significantly reduced lung metastatic foci formation in an i.v. model, and such inhibition was transiently observed also in an orthotopic model. The antimetastatic effect was correlated with a significant increase of prostacyclin (PGI2) metabolite and reduction of endothelin-1, sE-selectin, sI-CAM, and TGF-β plasma levels as well as decreased V-CAM expression on the endothelium. Combination therapy decreased fibrinogen binding to the resting platelets at the early stage of tumor progression (day 14). However, at the later stages (days 21 and 28), the markers of platelet activation were detected (increased JON/A antibody bound, P-selectin level, binding of fibrinogen, and vWf). Decreased aggregation as well as a lower release of TGF-β were detected in platelets incubated ex vivo with compounds tested from metastatic breast cancer patients. Although combination therapy increases E-cadherin, the increase of N-cadherin and α-SMA in tumor tissue was also observed. The results showed that at the early stages of tumor progression, combined therapy with DETA/NO and clopidogrel improves vasoprotective and antiplatelet activity. However, in advanced tumors, some adverse effects toward platelet activation can be observed.

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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Objective: This analysis estimated the cost-effectiveness of intravitreal aflibercept injection(s) (IAI) for wet age-related macular degeneration (wAMD) compared with other treatments in Japan., Methods: This was a cost-utility analysis based on published data. A state-transition cohort model was constructed with six health states based on best-corrected visual acuity in the better-seeing eye. The cycle time was 4 weeks, and the time horizon was 12 years. The model compared IAI 2 mg every 8 weeks (2q8) for 2 years after three initial monthly injections, ranibizumab as needed, ranibizumab 0.5 mg every 4 weeks (0.5q4), pegaptanib sodium 0.3 mg every 6 weeks, verteporfin photodynamic therapy (PDT), and best supportive care, assumed to include medical management and monitoring, but no active therapy. Costs (expressed as Japanese yen [JPY]) and quality-adjusted life years (QALYs) gained were estimated for each treatment and discounted at 2.0%. Input data were obtained from clinical studies, the Japanese drug tariff and social insurance reimbursement schedule, and expert opinion. The analysis was conducted from the societal perspective, including medical costs as well as costs of blindness., Results: IAI 2q8 was dominant (i.e. more effective in terms of QALYs and less costly) to all other comparators (ranibizumab as needed, ranibizumab 0.5q4, pegaptanib sodium, PDT, and best supportive care), as shown by the incremental cost-utility ratio (i.e. cost per QALY gained)., Limitations: The strengths of the analysis include the wide range of comparators evaluated and the use of Japanese-specific utility data. The limitations include the use of one eye, inclusion of published data up to 2 years only, and assumptions on disease course over 5 years., Conclusions: IAI 2q8 was more effective in terms of QALYs and less costly compared with other treatments for wAMD in Japan.

It has been suggested that the left medial prefrontal cortex (LmPFC) has an inhibitory role in controlling the right mPFC (RmPFC), thereby reducing the deleterious effects of stressors on emotional states. Here, we investigated the effects on anxiety of bilateral or unilateral injections of NOC-9 [a nitric oxide (NO) donor] and cobalt chloride (CoCl2; a synaptic inhibitor) into the mPFC of mice exposed to the elevated plus-maze (Experiments 1 and 2). The effects of restraint or social defeat on anxiety in undrugged mice were recorded at 5 min or 24 h after exposure to the stress (Experiment 3). Experiment 4 investigated the effects of LmPFC injection of CoCl2 combined with restraint or social defeat on anxiety, which was recorded 24 h later. Although intra-RmPFC NOC-9 produced anxiogenesis, its injection into the LmPFC, or bilaterally, did not change anxiety. Intra-RmPFC or -LmPFC injection of CoCl2 produced anxiolytic- and anxiogenic-like effects, respectively. Both restraint and social defeat produced anxiogenesis at 5 min, but defeated mice did not display anxiety 24 h after the stress. Although intra-LmPFC CoCl2 did not change anxiety, which was recorded 24 h later in non-stressed mice, this synaptic inhibitor produced a clear, anxiogenic-like effect in defeated (but not restrained) mice. These results suggest that (i) nitrergic activation of the RmPFC increases anxiety, which in turn is inhibited by NO production within the LmPFC; (ii) neuronal inhibition of the RmPFC or LmPFC elicits anxiolysis and anxiogenesis, respectively; and (iii) inactivation of the LmPFC results in recrudescence of anxiety induced by social defeat stress., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Nitric oxide (NO), an endogenous gas that plays a versatile role in the physiological system, has the ability to increase the intestinal absorption of water-soluble compounds through the paracellular route. However, it remains unclear whether NO can enhance the absorption of hydrophobic drugs through the transcellular route. In this study, we examined the absorption-enhancing effect of NO on intestinal permeability of hydrophobic drugs in rat intestine. The pretreatment of rat gastrointestinal sacs with NOC7, a NO-releasing reagent, significantly increased the permeation of griseofulvin from mucosa to serosa in the sacs prepared from the duodenum, but not in those prepared from the other regions such as jejunum, ileum, and colon. The absorption-enhancing effect of NOC7 on the duodenal permeation varied depending on the hydrophobicity of the drugs used. Furthermore, NOC7 treatment was found to be apparently ineffective on the griseofulvin permeation in the duodenum pretreated with dithiothreitol (DTT) that was used as a mucus remover, even though the permeation was increased by pretreatment with DTT alone. These results suggest that NO increases the absorption of hydrophobic drugs through the transcellular route in the duodenum by modulating the mucus layer function., (Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Background: DNAX accessory molecule-1 (DNAM-1) is an activating receptor constitutively expressed by macrophages/dendritic cells and by T lymphocytes and Natural Killer (NK) cells, having an important role in anticancer responses; in this regard, combination therapies able to enhance the expression of DNAM-1 ligands on tumor cells are of therapeutic interest. In this study, we investigated the effect of different nitric oxide (NO) donors on the expression of the DNAM-1 ligand Poliovirus Receptor/CD155 (PVR/CD155) in multiple myeloma (MM) cells., Methods: Six MM cell lines, SKO-007(J3), U266, OPM-2, RPMI-8226, ARK and LP1 were used to investigate the activity of different nitric oxide donors [DETA-NO and the NO-releasing prodrugs NCX4040 (NO-aspirin) and JS-K] on the expression of PVR/CD155, using Flow Cytometry and Real-Time PCR. Western-blot and specific inhibitors were employed to investigate the role of soluble guanylyl cyclase/cGMP and activation of the DNA damage response (DDR)., Results: Our results indicate that increased levels of nitric oxide can upregulate PVR/CD155 cell surface and mRNA expression in MM cells; in addition, exposure to nitric oxide donors renders myeloma cells more efficient to activate NK cell degranulation and enhances their ability to trigger NK cell-mediated cytotoxicity. We found that activation of the soluble guanylyl cyclase and increased cGMP concentrations by nitric oxide is not involved in the up-regulation of ligand expression. On the contrary, treatment of MM cells with nitric oxide donors correlated with the activation of a DNA damage response pathway and inhibition of the ATM /ATR/Chk1/2 kinase activities by specific inhibitors significantly abrogates up-regulation., Conclusions: The present study provides evidence that regulation of the PVR/CD155 DNAM-1 ligand expression by nitric oxide may represent an additional immune-mediated mechanism and supports the anti-myeloma activity of nitric oxide donors.

The dimensions of local flaps are often limited by the vascular supply to the distal aspect of the flap. Distal flap necrosis occurs if the vascular supply is inadequate. The purpose of this study was to investigate the use of iontophoretic delivery of nitric oxide (NO) donors to a local skin flap model to improve the survival area of the flap. Thirty-two male Sprague-Dawley rats (300 g) were divided into seven experimental groups to determine the effect of iontophoretic delivery of NO on surface perfusion and flap survival area. A caudally based 3 x 11 cm dorsal skin flap was used to measure the effect of iontophoretic delivery of NO donors to a local skin flap to improve survival area of the flap. Iontophoretic delivery of the NO donors sodium nitroprusside (SNP) and diethylenetriamine NONOate (DETA-NO) resulted in a significant increase in survival area and surface perfusion when compared with sham controls. Iontophoretic delivery of saline was associated with a 13% improvement in flap survival when compared with nontreated controls. Iontophoretic delivery and subcutaneous injection of NO donors (SNP and DETA-NO) increased skin flap viability by demonstrating improved flap survival areas. The results of this study suggest that NO may serve as a postoperative treatment of skin flaps to encourage skin flap survival and prevent distal necrosis.

Objective: Diethylenetriamine/nitric oxide (DETA/NO) has been shown to be an effective treatment for delayed posthemorrhagic vasospasm when released abluminally from ethylene-vinyl acetate copolymer (EVAc). However, the observed mortality associated with this drug warrants further investigation. To establish a maximum tolerable dose, this study evaluated the toxicity of DETA/NO released from EVAc in a dose-escalation series in cynomolgus monkeys (Macaca fascicularis)., Methods: DETA/NO was incorporated into EVAc at a 20:80 dry weight ratio (DETA/NO:EVAc). A total of 13 animals underwent a right frontotemporal craniotomy for placement of a single polymer delivering no drug (n = 3), 0.5 +/- 0.1 mg/kg (n = 3), 0.9 +/- 0.1 mg/kg (n = 3), 1.9 +/- 0.2 mg/kg (n = 3), or a 3.2 mg/kg dose (n = 1) into the subarachnoid space., Results: The animal receiving the highest dose of DETA/NO (3.2 mg/kg) died 46 hours after surgery. The remaining animals survived for the planned duration of the study. One animal in the group receiving the 1.9 mg/kg dose experienced a seizure 25 hours after surgery and remained lethargic for 2 days before making a complete recovery. The remaining animals exhibited no adverse behavioral effects. Histopathological examination of brain tissue revealed hemorrhagic and ischemic changes at doses above 0.9 mg/kg. No evidence of vascular wall pathology or infection was observed in any animal., Conclusion: The greatest amount of DETA/NO safely delivered from EVAc copolymer to the subarachnoid space of the cynomolgus monkey is approximately 1.0 mg/kg. These findings show that continuous intracisternal delivery of DETA/NO is a safe and potentially effective strategy for prophylactic treatment of delayed cerebral vasospasm.

The purpose of this study was to assess intrinsic smooth muscle mechanisms contributing to greater nitric oxide (NO) responsiveness in pulmonary vascular vs. airway smooth muscle. Canine pulmonary artery smooth muscle (PASM) and tracheal smooth muscle (TSM) strips were used to perform concentration response studies to an NO donor, (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO). PASM exhibited a greater NO responsiveness whether PASM and TSM were contracted with receptor agonists, phenylephrine and acetylcholine, respectively, or with KCl. The >10-fold difference in NO sensitivity in PASM was observed with both submaximal and maximal contractions. This difference in NO responsiveness was not due to differences in endothelial or epithelial barriers, since these were removed, nor was it due to the presence of cGMP-independent NO-mediated relaxation in either tissue. At equal concentrations of NO, the intracellular cGMP concentration ([cGMP]i) was also greater in PASM than in TSM. Phosphodiesterase (PDE) inhibition using isobutylmethylxanthine indicated that the greater [cGMP]i in PASM was not due to greater PDE activity in TSM. Expression of soluble guanylate cyclase (sGC) subunit mRNA (2 +/- 0.2 and 1.3 +/- 0.2 attomol/microg total RNA, respectively) and protein (47.4 +/- 2 and 27.8 +/- 3.9 ng/mg soluble homogenate protein, respectively) was greater in PASM than in TSM. sGCalpha1 and sGCbeta1 mRNA expression was equal in PASM but was significantly different in TSM, suggesting independent regulation of their expression. An intrinsic smooth muscle mechanism accounting for greater NO responsiveness in PASM vs. TSM is greater sGC activity.

The National Toxicology Program (NTP) is responsible for providing comprehensive toxicology evaluations of substances, while at the same time incorporating approaches to reduce, refine or replace laboratory animals in routine toxicity/carcinogenicity studies. Consistent with this, a series of metabolism studies in rodents and human liver slices, electron spin resonance spectroscopy (ESR) studies, short-term dermal toxicity studies in rodents, and acute bone marrow micronucleus studies in mice were performed on diazoaminobenzene (DAAB, also known as 1,3-diphenyltriazine). These studies demonstrated that DAAB is metabolized and shares similar genotoxic and toxicological properties to the known human carcinogen, benzene, and the known rodent carcinogen, aniline. These data were used to evaluate the potential carcinogenicity of DAAB without doing a 2-year rodent bioassay. Based on this analysis, DAAB was predicted to be carcinogenic if evaluated in a 2-year rodent bioassay. These data were evaluated to support listing DAAB in the NTP Report on Carcinogens as a substance 'reasonably anticipated to be a human carcinogen'. The purpose of this article is to review the data developed for predicting the carcinogenicity of DAAB., (Published in 2005 by John Wiley & Sons, Ltd.)

Object: Results of prior studies in rats and rabbits show that the alteration of vasomotor tone in vasospasm following periadventitial blood exposure may be reversed, at least in part, by the administration of compounds releasing nitric oxide (NO). The authors have now generalized this finding to nonhuman primates., Methods: Ten cynomolgus monkeys underwent cerebral angiography before and 7 days following the induction of subarachnoid hemorrhage (SAH) by the placement of 2 to 3 ml clotted autologous blood around the supraclinoid carotid, proximal anterior cerebral, and proximal middle cerebral arteries. An ethylene vinyl acetate copolymer, either blank (five animals) or containing 20% w/w (Z)-1-[2-(2-aminoethyl)-N-(2-aminoethyl)amino]diazen-1-ium-1,2-diolate (DETA/NO, 4.3 mg/kg; five animals) was placed adjacent to the vessels at the time of surgery. Animals were killed on Day 7 post-SAH following repeated cerebral angiography. The mean percentage of control vascular areal fraction was calculated from angiograms. Cerebral vessels were sectioned and the mean percentage of lumen patency was calculated. One animal that had received the DETA/NO polymer died prior to repeated angiography. In the remaining animals, DETA/NO caused a significant decrease in vasospasm compared with controls, according to both angiographic (84.8 +/- 8.6 compared with 56.6 +/- 5.2%, respectively, p < 0.05) and histological studies (internal carotid artery 99.3 +/- 1.8 compared with 60.1 +/- 4.4%, respectively, p < 0.001; middle cerebral artery 98.4 +/- 3 compared with 56.1 +/- 3.7%, respectively, p < 0.001; and anterior cerebral artery 89.2 +/- 8.5 compared with 55.8 +/- 6.3%, respectively, p < 0.05)., Conclusions: The controlled release of DETA/NO is effective in preventing delayed cerebral vasospasm in an SAH model in nonhuman primates. The death of one animal in the treatment group indicates that the present dosage is at the threshold between therapeutic efficacy and toxicity.

Objective: Nitric oxide (NO) has a toxic effect on neuronal cells related to glutamate receptors. NO released from post-synaptic cells with glutamate receptors can induce nearby cell death. In this experiment, we examined the effect of NO on cochlear hair cells., Methods: Two concentrations, 0.5 and 0.1 molar concentration (MC) of 1-hydroxy-2-oxo-3-(3-aminopropyl)-3-isopropyl-1-triazene (NOC-5), which is a NO donor, were placed on the round window of the guinea pig cochlea and its morphological changes were investigated at 12, 28, 72 h after the treatment., Results: By the trypan blue dye extrusion method, the cell death was recognized in the outer hair cells at the percentage 0, 36.0+/-6.6, 4.9+/-1.2% at 12, 28, 72 h after treatment respectively in the group 0.5 MC NOC-5, but no inner hair cell death was recognized. In the group of 0.1 MC NOC-5 and control any cell death was not detected. Transmission electron microscopy revealed this cell death was characteristic of apoptosis., Conclusion: The findings that apoptosis was induced in the outer hair cells of the organ of Corti by NO suggests the possibility that the cochlea is affected when extra NO release occurs there, leading to cochlear dysfunction.

The synthesis and abl tyrosine kinase inhibitory activities of alkyltriazenes conjugated to phenylaminopyrimidines are described. Significant abl inhibitory activities were observed only when a benzamido spacer was inserted between the 1,2,3-triazene chain and the 2-phenyaminopyridopyrimidine moiety.

Alkylating agents are widely used in high-dose chemotherapy regimens in combination with hematological support. Knowledge about the pharmacokinetics and pharmacodynamics of these agents administered in high doses is critical for the safe and efficient use of these regimens. The aim of this review is to summarize the clinical pharmacology of the alkylating agents (including the platinum compounds) in high-dose chemotherapy. Differences between conventional and high doses will be discussed.

Object: Intrathecal bolus administration of (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)aminio]diazen++ +-1-ium-1,2-diolate (DETA/NO), a long half-life diazeniumdiolate-class nitric oxide (NO) donor, was evaluated for safety and efficacy in the treatment of delayed cerebral vasospasm in a canine model of subarachnoid hemorrhage (SAH)., Methods: The baseline basilar artery (BA) diameter of 25 dogs was measured with the aid of angiography on Day 0. Vasospasm was then induced by intracisternal injection of autologous arterial blood on Days 0 and 2. Repeated arteriography on Day 7 revealed an average BA diameter of 58% of baseline. Each dog was then randomized to one of four groups: a pathology control group (SAH only, four animals); a treatment control group (SAH plus 2 micromol of the inactive drug carrier DETA, eight animals); a low-dose treatment group (SAH plus 0.2 micromol DETA/NO, six animals); or a high-dose treatment group (SAH plus 2 micromol DETA/NO, six animals). The drugs were administered in a 2-ml intrathecal bolus via the cisterna magna. Arterial caliber was monitored by angiography over the subsequent 4 hours. A 2-micromol dose of the drug was then given and serial arteriography continued for an additional hour to screen for tachyphylaxis. Intracranial pressure and respiratory and hemodynamic parameters were continuously monitored. Histopathological analyses of the animals' brains were performed after the dogs were killed on Day 8. The drug DETA/NO produced reversal of vasospasm in a dose-dependent fashion that roughly followed a double exponential time course. Doses of 2 micromol DETA/NO resulted in restoration of the angiographically monitored BA diameter to the prevasospasm size at 1.5 hours posttreatment, and this was sustained at 88% of baseline at 4 hours (p < 0.01, independent samples t-test). By contrast, the treatment control group remained on average at 54% of baseline diameter. The low-dose treatment group achieved only partial and more transitory relaxation. Histopathological analyses showed findings consistent with chronic SAH but did not demonstrate any toxicity associated with the NO donor. No adverse physiological changes were seen., Conclusions: This study indicates that long-acting NO donors are potentially useful as agents to restore circulation in patients suffering from cerebral vasospasm.

Adverse physiological effects can often interfere with the use of nitric oxide (NO) as a therapeutic agent, especially when it is systemically generated from prodrugs. NO which is generated and delivered site-specifically by transdermal donors may be useful in the treatment of parasitic, bacterial or viral skin infections without causing systemic side effects. Three diazeniumdiolates (formerly "NONOate"), including two water soluble compounds, (Z)-1-[N-2-aminoethyl)-N-(2-ammonioethyl)amino]-diazen-1- ium-1,2-diolate (DETA-NO) and (Z)-1-[N-(3-aminopropyl)-N-(3-ammoniopropyl)amino] diazen-1-ium-1,2-diolate (DPTA-NO), and one insoluble compound, DPTA-NO grafted to dextran microspheres (DPTA-NO-g-dextran) were used to transdermally deliver NO to rats. Dextran microspheres were obtained by simultaneously grafting DPTA-NO to dextran and cross-linking dextran with CNBr in an oil-water emulsion. Suspended in hydrogel, DETA-NO, DPTA-NO, and DPTA-NO-g-dextran were applied three times to depilated rats at 4 day intervals. Results show that metabolic urinary nitrate levels increase with time (24-48 h), reach a maximum, and return to baseline by the fourth day. DPTA-NO applications produced an average maximum nitrate level of 94.2 mumol/day +/- 34.2 mumol S.D. compared to the average maximum nitrate level of 39.8 mumol/day +/- 8.6 mumol S.D. obtained from treatment with DETA-NO. These results suggest that DPTA-NO delivered NO more efficiently than DETA-NO. When DPTA-NO-g-dextran microspheres were used as the NO donor, results comparable to DPTA-NO were observed with an average maximum nitrate level of 14.9 mumol/day +/- 3.0 mumol S.D. These and other conclusive data indicate that, via these diazeniumdiolates, NO can be delivered effectively through rat skin.

High levels of O6-alkylguanine-DNA-alkyltransferase (OGAT) can, at least in part, account for tumor cell resistance to O6-alkylguanine alkylating agents, including triazene compounds. A pilot clinical study indicates that dacarbazine can induce a marked decrease of leukemic blasts in patients affected by acute myelogenous leukemia (AML) with low pretreatment levels of OGAT activity. In this study we show a synergistic antitumor effect between cisplatin (CDDP) and temozolomide (an in vitro active analog of dacarbazine), following combined in vitro treatment of leukemic blasts. Synergistic effect appears to be CDDP-dose dependent. In vivo treatment of leukemic patients with CDDP was followed by a reduction of OGAT activity in 2 out 3 cases. These data point out that CDDP could be a good candidate for depleting OGAT protein of leukemic cells, thus reversing tumor cell resistance to dacarbazine.

The antitumor and antimetastatic effects of p-(3-methyl-1-triazeno)benzoic acid potassium salt (MM-COOK) as compared with those of the parent 3,3-dimethyl derivative (DM-COOK) were examined using Lewis lung carcinoma, MCa mammary carcinoma of the CBA mouse and TLX5 lymphoma. Similarly to DM-COOK, MM-COOK reduces metastasis formation and significantly prolongs the survival of mice bearing the Lewis lung carcinoma when given at a daily dose corresponding to one-half that of DM-COOK. Unlike DM-COOK, MM-COOK exhibits significant cytotoxicity to metastatic foci and pronounced inhibition of primary tumor development. MM-COOK also causes cytotoxic effects on TLX5 lymphoma cell growing in the peritoneal cavity, even when used at low doses. The antimetastatic effects observed in mice bearing MCa mammary carcinoma are unrelated to the inhibition of primary tumor growth and are more likely due to the selection of clones endowed with lower metastatic ability. It appears that MM-COOK exhibits the same antineoplastic activity as DM-COOK, but the former does so at a lower daily dose and produces interesting cytotoxic effects other than those reflecting its antimetastatic properties. It thus seems to be a valid alternative to DM-COOK, in view of the possible introduction of newer aryltriazenes into clinical use.

The effects of 5-(3,3-Dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC), 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea (MeCCNU), and L-phenylalanine mustard (L-PAM) have been compared by using three i.p. transplanted mouse melanomas: the B16 melanoma in C57BL/6 mice; the Harding-Passey (HP) malanoma in BALB/c X DBA/2F1 (hereafter called CD2F1) mice; and the Cloudman S91 melanoma in DBA/2 mice. HP melanoma responds well to all three drugs. S91 responds only to L-PAM and MeCCNU. DTIC may accelerate death in mice bearing this tumor. B16 responds well to L-PAM and moderately well to MeCCNU and to multiple injections of DTIC. The best response to DTIC and MeCCNU is given by HP, while the best response to L-PAM is given by S91. Tumor cell-doubling times were found to be 1.5 days for B16, 2 DAYS FOR HP, and 3 days for S91. HP would seem to be the most responsive malanmoma with respect to the 3 agents studied. This may be due to an interaction between the chemotherapeutic agents and the host immune response, since the HP tumor arose in a noninbred mouse and is thus nonsyngeneic with the CD2F1 host. All three tumors appear to be interesting biological models for studying drug combinations and combined therapeutic modalities against melanoma.

Chemotherapy for metastatic melanoma was performed in 80 consecutive evaluable patients. DTIC, BCNU and CCNU produced responses in 28% of patients, alone or in combination with each other. Three of 62 patients treated with DTIC remain free of tumor, off therapy at 18-36 months following objective regression of metastases. Chemotherapy with commercially available drugs continued to be uniformly unsuccessful. DTIC was used successfully in treatment of extensive extracranial disease, including one patient with metastatic melanoma during pregnancy. Cerebral metastases were the sole or major cause of death in 8/9 patients who relapsed following control with DTIC for nine months or longer, and one patient developed a carcinoma of the breast following therapy with DTIC and BCNU. Remission was induced in two patients with intralesional BCG, after prior attempts to control metastases with DTIC and combination chemotherapy.

Six intracarotid artery infusions of DTIC were performed in 4 patients with invasive malignant melanoma of the maxillo-facial region. A total dose of 3.5 to 7 g was well tolerated. Only 1 patient who received 9.5 g in 25 days developed a reversible bone marrow depletion. Two out of the 4 patients presented a transient 50% tumor surface regression, but no response was seen after the second infusion. Of the 2 remaining patients, 1 had no measurable effect and 1 experienced tumor progression.

1-Phenyl- and 1-(pyridyl-3)-3,3-diethyltriazene have been investigated for carcinogenic effects in BD-rats after subcutaneous injection of a single dose of 50 mg of the compounds on day 1, 10, or 30 of life. The phenyl derivative showed a lower carcinogenic activity in comparison with the pyridyltriazene. Predominantly neurogenic tumors were observed. These were seen much more on treatment on day 1 or 10 of life than on treatment on day 30. The results confirm that the nervous system of rats is a target organ predominantly during early postnatal life.

Two anticancer agents, one lipophilic, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), and one hydrophilic, 5-(3,3-dimethyl-1-triazeno) imidazol-4-carboxamide (DTIC), were used to treat Brown-Pearce epithelioma in the anterior chamber of rabbit eyes. The BCNU test animals were divided into three groups: one treated by direct injection of the drug into the subconjuntival space or the anterior chamber, the second by both direct injections and intravenous administration, and the third by intravenous injection alone. The DTIC test animals were treated with only local injection into the subconjunctival space or anterior chamber. Dosage, delivery system, and effectiveness were compared following clinical observation and histopathologic examination. Direct delivery of BCNU or DTIC in subconjuntival space or anterior chamber delayed the growth of Brown-Pearce epithelioma in rabbit eye. The effectiveness of this treatment was significnatly enhanced by combining direct injection with systemic administration of a lower dose of BCNU.

D.T.I.C. appears more and more to possess the characteristics of an effective chemical agent in the treatment of naevocarcinoma. Having been able to include it in a mixed chemotherapy programme used in a group of 44 patients, we report here our findings. This is a preliminary study, the results of which will have to be confirmed in time on a larger group of patients over a longer period of observation.

The therapeutic efficacy of the preoperative administration of DTIC and its benzenoid analog DM-COOK, in terms of prolongation of the life-span of mice bearing Lewis lung carcinoma lines with different metastatic potential, has been evaluated in combined experiments with surgery and postsurgical non-specific immunotherapy with the peptidoglycan monomer PGM. The effects of the presurgical selective antimetastatic treatment with the dimethyltriazenes consist of a statistically significant prolongation of the survival time of the treated mice. The activity is more pronounced, in terms of animals cured, using the tumor line which is endowed of a low ability to colonize the lungs. The addition of postsurgical immunotherapy with PGM improves the overall therapeutic efficacy of the combined treatment with triazenes and surgery, independently from the tumor line used. In general, the presurgical treatment with the benzenoid triazene DM-COOK, causes antineoplastic effects slightly better than those observed using the imidazole derivative DTIC.

The effects of treatment by immunotherapy and chemoimmunotherapy were assessed in 31 non-randomized patients with melanoma; 16 received only BCG and 15 BCG and chemotherapy. For advanced cases, the times of survival for the two treatment groups did not differ significantly, nor did there appear to be any improvement in survival over that recorded for the natural course of the disease. The pretreatment immune status as judged by hyporeactivity or normal reactivity to skin tests for delayed type hypersensitivity did not appear to influence survival. This study, together with other reports, does not support the use of immunotherapy in advanced melanoma. Immunotherapy for early melanoma is still to be assessed.

Adding duborimycin to the association of vincristine + DTIC in the treatment of malignant melanoma produces therapeutic results interesting but not statistically significant and not counter-balancing the toxic side effects. This study confirms the lack of positive results of polychemotherapy in melanoma as compared to treatment by an efficacious single drug therapy (DTIC); it also emphasizes the necessity of randomized studies so that valuable comparisons may be done.

1-p-(3,3-dimethyl-1-triazeno) benzoic acid potassium salt (DM-COOK) was tested on M 5076/73A (M5) mouse sarcoma at a dose of 40 or 50 mg/kg/day (Days 6--14) after im transplant of 7 x 10(5) cells, with or without surgical removal of the primary tumor on Day 14. Treatment at either dose level resulted in reduction of the primary tumor weight to around 50% of that in the controls, and striking antimetastatic effects were observed. When a dose of 40 or 50 mg/kg of DM-COOK was followed by surgery, there were 14% and 40% long-term survivors, respectively, but the higher dose caused about 30% toxic deaths. After iv injection of 10(3) or 10(5) M5 tumor cells, no artificial metastases appeared in DM-COOK-treated mice, whereas all control animals had metastatic involvement in the liver, spleen, ovaries, and kidneys.

A group of 20 male F344 rats was given by gavage a solution of 2.5 mg of 1,3-diethyltriazene in 0.2 ml corn oil twice a week for 20 weeks. Diethyltriazene is very unstable in hydroxylic solvents, but when administered in oil by gavage it induced tumors in almost all of the treated animals, 16 of which had neoplasms of the forestomach; 13 of which were carcinomas. In addition 9 rats had adenomas of the nasal mucosa, two had alveolar-bronchiolar adenomas of the lung, one had a carcinoma of the esophagus and one an adenocarcinoma of the ileum. Half of the rats had died by week 56 of the experiment, the first at week 40 and the last at week 73. The induction of tumors at sites distant from the stomach indicate that diethyltriazene is more stable in the body than would be predicted from its chemical behavior. Although diethyltriazene is a protected alkyldiazonium ion, its carcinogenic effects in rats differ from ethylnitrosoureas, which are also ethylating agents.