A Novel Dual-Function Nitric Oxide Donor Therapy for Preeclampsia-A Proof-of-Principle Study in a Murine Model.

Background: Preeclampsia (PE) is a hypertensive disorder of pregnancy that is associated with substantial morbidity and mortality for the mother and fetus. Reduced nitric oxide bioavailability and oxidative stress contribute to the maternal and fetal pathophysiology of PE. In this study, we evaluated the efficacy of a novel dual-function nitric oxide donor/redox modulator, AKT-1005, in reducing PE symptoms in a mouse model of PE. Method: The potential therapeutic effect of AKT-1005 was tested in an animal model of Ad.sFlt-1-induced hypertension, proteinuria and glomerular endotheliosis, a model of PE. Pregnant Ad.sFlt-1-overexpressing CD1 mice were randomized into groups administered AKT-1005 (20 mg/kg) or a vehicle using a minipump on gd11 of pregnancy, and the impact on blood pressure and renal and placental damage were assessed. Results: In healthy female mice, ex vivo treatment of resistance vessels with AKT-1005 induced vasorelaxation, and 6 days of treatment in vivo did not significantly alter blood pressure with or without pregnancy. When given for 6 days during pregnancy along with Ad.sFlt-1-induced PE, AKT-1005 significantly increased plasma nitrate levels and reduced hypertension, renal endotheliosis and plasma cystatin C. In the placenta, AKT-1005 improved placental function, with reduced oxidative stress and increased endothelial angiogenesis, as measured by CD31 staining. As such, AKT-1005 treatment attenuated the Ad.sFlt-1-induced increase in placental and free plasma soluble endoglin expression. Conclusions: These data suggest that AKT-1005 significantly attenuates the sFlt-1-induced PE phenotypes by inhibiting oxidative stress, the anti-angiogenic response, and increasing NO bioavailability. Additional research is warranted to investigate the role of AKT-1005 as a novel therapeutic agent for vascular disorders such as preeclampsia.

A Novel Dual-Function Redox Modulator Relieves Oxidative Stress and Anti-Angiogenic Response in Placental Villus Explant Exposed to Hypoxia-Relevance for Preeclampsia Therapy.

BACKGROUND: Preeclampsia (PE) is a severe, life-threatening complication during pregnancy (~5-7%), and no causative treatment is available. Early aberrant spiral artery remodeling is associated with placental stress and the release of oxygen radicals and other reactive oxygen species (ROS) in the placenta. This precedes the production of anti-angiogenic factors, which ultimately leads to endothelial and trophoblast damage and the key features of PE. We tested whether a novel dual-function redox modulator-AKT-1005-can effectively reduce placental oxidative stress and alleviate PE symptoms in vitro. METHOD: Isolated human villous explants were exposed to hypoxia and assessed to determine whether improving cell-redox function with AKT-1005 diminished ROS production, mitochondrial stress, production of the transcription factor HIF1A, and downstream anti-angiogenic responses (i.e., sFLT1, sEng production). MitoTEMPO was used as a reference antioxidant. RESULTS: In our villous explant assays, pretreatment with AKT-1005 reduced mitochondrial-derived ROS production, reduced HIF-1A, sFLT1, and sEng protein expression, while increasing VEGF in hypoxia-exposed villous trophoblast cells, with better efficiency than MitoTEMPO. In addition, AKT-1005 improved mitochondrial electron chain enzyme activity in the stressed explant culture. CONCLUSIONS: The redox modulator AKT-1005 has the potential to intervene with oxidative stress and can be efficacious for PE therapy. Future studies are underway to assess the in vivo efficacy of HMP.

Modulation of oxidative and nitrosative stress attenuates microvascular hyperpermeability in ovine model of Pseudomonas aeruginosa sepsis.

In sepsis, microvascular hyperpermeability caused by oxidative/nitrosative stress (O&NS) plays an important role in tissue edema leading to multi-organ dysfunctions and increased mortality. We hypothesized that a novel compound R-107, a modulator of O&NS, effectively ameliorates the severity of microvascular hyperpermeability and preserves multi-organ function in ovine sepsis model. Sepsis was induced in twenty-two adult female Merino sheep by intravenous infusion of Pseudomonas aeruginosa (PA) (1 × 1010 CFUs). The animals were allocated into: 1) Control (n = 13): intramuscular injection (IM) of saline; and 2) Treatment (n = 9): IM of 50 mg/kg R-107. The treatment was given after the PA injection, and monitored for 24-h. R-107 treatment significantly reduced fluid requirement (15-24 h, P < 0.05), net fluid balance (9-24 h, P < 0.05), and water content in lung/heart/kidney (P = 0.02/0.04/0.01) compared to control. R-107 treatment significantly decreased lung injury score/modified sheep SOFA score at 24-h (P = 0.01/0.04), significantly lowered arterial lactate (21-24 h, P < 0.05), shed syndecan-1 (3-6 h, P < 0.05), interleukin-6 (6-12 h, P < 0.05) levels in plasma, and significantly attenuated lung tissue 3-nitrotyrosine and vascular endothelial growth factor-A expressions (P = 0.03/0.002) compared to control. There was no adverse effect in R-107 treatment. In conclusion, modulation of O&NS by R-107 reduced hyperpermeability markers and improved multi-organ function.

The peroxynitrite catalyst WW-85 improves pulmonary function in ovine septic shock.

Systemic inflammatory response syndrome is associated with excessive production of nitric oxide (NO·) and superoxide (O2), forming peroxynitrite, which in turn, acts as a terminal mediator of cellular injury by producing cell necrosis and apoptosis. We examined the effect of the peroxynitrite decomposition catalyst, WW-85, in a sheep model of acute lung injury and septic shock. Eighteen sheep were operatively prepared and randomly allocated to the sham, control, or WW-85 group (n = 6 each). After a tracheotomy, acute lung injury was produced in the control and WW-85 groups by insufflation of four sets of 12 breaths of cotton smoke. Then, a 30-mL suspension of live Pseudomonas aeruginosa bacteria (containing 2 - 5 × 10¹¹ colony-forming units) was instilled into the lungs according to an established protocol. The sham group received only the vehicle (30 mL saline). The sheep were studied in awake state for 24 h and ventilated with 100% oxygen. WW-85 was administered 1 h after injury as bolus infusion (0.1 mg/kg), followed by a continuous infusion of 0.02 mg·kg⁻¹·h⁻¹ until the end of the 24-h experimental period. Compared with injured but untreated controls, WW-85-treated animals had significantly improved gas exchange, reductions in airway obstruction, shunt formation, lung myeloperoxidase concentrations, lung malondialdehyde concentrations, lung 3-nitrotyrosine concentrations, and plasma nitrate-to-nitrite levels. Animals treated with WW-85 exhibited less microvascular leakage and improvements in pulmonary function. These results provide evidence that blockade of the nitric oxide-peroxynitrite pathway improves disturbances from septic shock, as demonstrated in a clinically relevant ovine experimental model.

The novel inosine analogue INO-2002 exerts an anti-inflammatory effect in a murine model of acute lung injury.

Endogenous purines, including inosine, have been shown to exert immunomodulatory and anti-inflammatory effects in a variety of disease models. The dosage of inosine required for these effects has been shown to be between 200 and 600 mg kg(-1) because of the rapid metabolism of inosine in vivo. The aim of this study was to determine whether a metabolic resistant purine analog, INO-2002, exerts anti-inflammatory effects in an animal model of acute respiratory distress syndrome. Mice challenged with intratracheal LPS (50 microg) were treated with INO-2002 (30 or 100 mg kg(-1), i.p.) in divided doses at either 1 and 12 h or at 5 and 16 h. After 24 h, bronchoalveolar lavage fluid was obtained to measure leukocyte infiltration by myeloperoxidase levels, lung edema by protein levels, and proinflammatory chemokine (macrophage inflammatory protein 1alpha) and cytokine (TNF-alpha, IL-1, and IL-6) levels. INO-2002 (30 and 100 mg kg(-1)) reduced the LPS-mediated infiltration of leukocytes and edema as evidenced by bronchoalveolar lavage fluid reduction in levels of myeloperoxidase and protein. INO-2002 also downregulated expression of the proinflammatory mediators macrophage inflammatory protein 1alpha, TNF-alpha, IL-1, and IL-6. Delaying the start of treatment by 5 h after LPS administration affected the potency of INO-2002 protective effects, with 100 but not 30 mg kg(-1) having anti-inflammatory effects. The inosine analog INO-2002 largely suppressed LPS-induced inflammation in vivo at doses lower than those needed for the naturally occurring purine inosine. These data support the proposal that purine analogs, resistant to metabolic breakdown, may represent a useful addition to the therapy of acute respiratory distress syndrome.

Synthesis of (R)-3,4-dihydro-2H-pyran-2-carboxaldehyde: application to the synthesis of potent adenosine A(2A) and A(3) receptor agonist.

Synthesis of potent adenosine A(2A) and A(3) receptor agonist from the modification of adenosine-5'-N-ethylcarboxamide (NECA) has been reported. Diastereoisomer possessing an (R) 3,4-dihydro-2H-pyranyl (DHP) moiety exhibited the highest affinity at the A(2A) and A(3) receptors. The key steps involve the synthesis of (R)-3,4-dihydro-2H-pyran-2-carboxaldehyde (7), which was obtained through the enzyme catalyzed kinetic resolution of (±)-2-acetoxymethyl-3,4-dihydro-2H-pyran (5).

The novel inosine analogue, INO-2002, protects against diabetes development in multiple low-dose streptozotocin and non-obese diabetic mouse models of type I diabetes.

Endogenous purines including inosine have been shown to exert immunomodulatory and anti-inflammatory effects in a variety of disease models. The dosage of inosine required for protection is very high because of the rapid metabolism of inosine in vivo. The aim of this study was to determine whether a metabolic-resistant purine analogue, INO-2002, exerts anti-inflammatory effects in two animal models of type I diabetes. Type I diabetes was induced chemically with streptozotocin or genetically using the non-obese diabetic (NOD) female mouse model. Mice were treated with INO-2002 or inosine as required at 30, 100, or 200 mg/kg per day, while blood glucose and diabetes incidence were monitored. The effect of INO-2002 on the pancreatic cytokine profile was also determined. INO-2002 reduced both the hyperglycaemia and incidence of diabetes in both streptozotocin-induced and spontaneous diabetes in NOD mice. INO-2002 proved to be more effective in protecting against diabetes than the naturally occurring purine, inosine, when administered at the same dose. INO-2002 treatment decreased pancreatic levels of interleukin (IL)-12 and tumour necrosis factor-alpha, while increasing levels of IL-4 and IL-10. INO-2002 also reduced pancreatic levels of the chemokine MIP-1 alpha. The inosine analogue, INO-2002, was protected more effectively than the naturally occurring purine, inosine, against development of diabetes in two separate animal models. INO-2002 exerts protective effects by changing the pancreatic cytokine expression from a destructive Th1 to a protective Th2 profile. The use of analogues of inosine such as INO-2002 should be considered as a potential preventative therapy in individuals susceptible to developing type I diabetes.

Nitrative inactivation of thioredoxin-1 and its role in postischemic myocardial apoptosis.

BACKGROUND: Intracellular proteins involved in oxidative stress and apoptosis are nitrated in diseased tissues but not in normal tissues; definitive evidence to support a causative link between a specific protein that is nitratively modified with tissue injury in a specific disease is limited, however. The aims of the present study were to determine whether thioredoxin (Trx), a novel antioxidant and antiapoptotic molecule, is susceptible to nitrative inactivation and to establish a causative link between Trx nitration and postischemic myocardial apoptosis. METHODS AND RESULTS: In vitro exposure of human Trx-1 to 3-morpholinosydnonimine resulted in significant Trx-1 nitration and almost abolished Trx-1 activity. 3-morpholinosydnonimine-induced nitrative Trx-1 inactivation was completely blocked by MnTE-2-PyP(5+) (a superoxide dismutase mimetic) and markedly attenuated by PTIO (a nitric oxide scavenger). Administration of either reduced or oxidized Trx-1 in vivo attenuated myocardial ischemia/reperfusion injury (>50% reduction in apoptosis and infarct size, P<0.01). However, administration of nitrated Trx-1 failed to exert a cardioprotective effect. In cardiac tissues obtained from ischemic/reperfused heart, significant Trx-1 nitration was detected, Trx activity was markedly inhibited, Trx-1/ASK1 (apoptosis signal-regulating kinase-1) complex formation was abolished, and apoptosis signal-regulating kinase-1 activity was increased. Treatment with either FP15 (a peroxynitrite decomposition catalyst) or MnTE-2-PyP(5+) 10 minutes before reperfusion blocked nitrative Trx inactivation, attenuated apoptosis signal-regulating kinase-1 activation, and reduced postischemic myocardial apoptosis. CONCLUSIONS: These results strongly suggest that nitrative inactivation of Trx plays a proapoptotic role under those pathological conditions in which production of reactive nitrogen species is increased and that antinitrating treatment may have therapeutic value in those diseases, such as myocardial ischemia/reperfusion, in which pathological apoptosis is increased.

Inhibition of experimental gingivitis in beagle dogs with topical mercaptoalkylguanidines.

BACKGROUND: Nitric oxide is a free radical produced in host tissues by constitutive and inducible forms of the enzyme nitric oxide synthase. Nitric oxide plays physiological roles, but it is also involved in the pathophysiology of several inflammatory conditions, including arthritis, ulcerative colitis, and circulatory shock. Local increases in inducible nitric oxide synthase (iNOS) and reactive nitrogen products have also been demonstrated in humans and animals with periodontal disease. This masked, randomized, placebo-controlled preclinical investigation examined the effect of two mercaptoalkylguanidines, mercaptoethylguanidine (MEG) and guanidinoethyldisulfide (GED), which are iNOS inhibitors and reactive nitrogen scavenging compounds, on the development of experimental gingivitis in beagle dogs. METHODS: Fifteen female, 1-year-old beagles first completed a 2-week dose-escalation experiment during which a maximum tolerated dose was determined for MEG and GED gels. Thereafter, all animals were brought to optimal gingival health by mechanical scaling, followed by rigorous daily toothbrushing over a 4-week washout period. Experimental gingivitis was then induced, with cessation of plaque control and institution of a soft diet over 8 weeks. Beagles randomly received 0.3% MEG, 0.3% GED, or placebo (vehicle) gels, topically applied twice daily to premolar teeth. Gingival inflammation, bleeding tendency, and supragingival plaque were clinically measured at baseline and at 2, 3, 4, 6, and 8 weeks. Comparisons among groups and between group pairs (active versus placebo) were made using Kruskal-Wallis tests. RESULTS: From baseline to day 7, all groups expressed similar indices. Thereafter, significant and time-dependent increases in the plaque index (PI), gingival index (GI), and percentage of bleeding on probing (%BOP) were observed in placebo-treated beagles. Mean GI scores for beagles treated with GED or MEG gels remained at or below baseline levels for the entire treatment period. At weeks 2, 3, 4, and 8, GI scores were significantly lower for MEG and GED groups compared to the placebo group (P<0.05). In addition, MEG and GED gels significantly reduced gingival bleeding responses by 8 weeks (P<0.05). Although placebo-treated beagles demonstrated %BOP scores of 43% at week 8, GED- and MEG-treated beagles exhibited %BOP scores of 21% and 26%, respectively. Since no statistical difference among PI scores was noted for any of the time points, neither mercaptoalkylguanidine appeared to affect supragingival plaque levels. CONCLUSION: The data from this preclinical study indicate that mercaptoalkylguanidines, topically administered, may significantly reduce experimental gingivitis in the beagle dog.

Discovery of potent poly(ADP-ribose) polymerase-1 inhibitors from the modification of indeno[1,2-c]isoquinolinone.

Novel indeno[1,2-c]isoquinolinone derivatives were synthesized and evaluated as inhibitors of the nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1). These potent nonmutagenic PARP-1 inhibitors possess an additional five-membered ring between the B and C rings of 6(5H)-phenanthridinone. The most potent PARP-1 inhibitors were obtained from the substitution of the D ring at the C-9 position, in particular sulfonamide and N-acyl analogues (6 and 11). The 9-sulfonamide analogues 11a and 12a exhibited IC(50) values of 1 and 10 nM, respectively.

Facile and convenient syntheses of 6,11-dihydro-5H-indeno[1,2-c]isoquinolin- 5-ones and 6,11-dihydro-5H-indolo[3,2-c]isoquinolin-5-one.

[reaction: see text] The synthesis of 6,11-dihydro-5H-indeno[1,2-c]isoquinolin-5-ones from the base-promoted condensation reaction of homophthalic anhydride and 2-(bromomethyl)-benzonitrile and a convenient method for the synthesis of indolo[3,2-c]isoquinolinones are described.

Suppression of intestinal polyposis in Apcmin/+ mice by targeting the nitric oxide or poly(ADP-ribose) pathways.

Min mice have a germ-line nonsense mutation at codon 850 of the adenomatous polyposis coli (Apc) gene. These mice spontaneously develop multiple polyps in the small and large intestine at the age of 10-12 weeks. The aim of this study was to assess the role of reactive nitrogen species and poly(ADP-ribose) synthetase in tumorogenesis. Oxidative stress was found to be increased in the mucosa of the small intestine of Apc(min/+) mice with a concomitant increase in intestinal polyposis over control mice. Pharmacological inhibition of inducible nitric oxide synthase (NOS) with guanidinoethyldisulfide (GED) or stimulation of the breakdown of the nitrogen reactive species peroxynitrite using a potent decomposition catalyst, FP 15, reduced both the intestinal tumor load and the oxidative stress associated with intestinal polyposis in Apc(min/+) mice. Surprisingly, pharmacological inhibition of poly(ADP-ribose) synthetase by the phenanthridinone derivative PJ 34 also reduced the intestinal polyposis and oxidative stress in these mice, possibly through the inhibition of induction of nitric oxide synthase. These results suggest that reactive nitrogen species particularly peroxynitrite play a pivotal role in development of intestinal polyposis and that strategies to reduce both the oxidative stress and the formation of these radical species may be potential chemopreventive approaches for colorectal cancers.

Poly(ADP-ribose) polymerase inhibition protect neurons and the white matter and regulates the translocation of apoptosis-inducing factor in stroke.

Focal cerebral ischemia activates the nuclear protein poly(ADP-ribose) polymerase (PARP). Apoptosis-inducing factor (AIF) is a flavoprotein that is normally confined to the mitochondria, but translocates to the nucleus, as shown by in vitro models of neuronal injury. Using INO-1001, a novel potent inhibitor of PARP, we determined the role of PARP activation in the process of AIF translocation in a rat model of focal cerebral ischemia. The potency of INO-1001 as a PARP inhibitor and its cytoprotective potential in oxidant-challenged human neuronal SK-N-MC cells was first confirmed in vitro. PARP inhibition markedly reduced infarct size and improved neurological status in both transient and permanent models of MCA occlusion in Sprague-Dawley rats, with a therapeutic window of 6 h and 2 h in the transient and permanent ischemia models, respectively. The PARP inhibitor reduced the accumulation of poly(ADP-ribose) in the ischemic/reperfused hemisphere and reduced the accumulation of APP in the white matter of the affected hemisphere, consistently with protection against neuronal necrosis and axonal damage, respectively. Immunohistochemical analysis showed the appearance of AIF labeling in neuronal nuclei of the border zone ischemic area in the striatum after stroke. Cytoplasmatic (axonal) AIF staining was significantly diminished in the necrotic core of the striatum, while it was somewhat enhanced at the borderline ischemic territories of the white matter. Inhibition of PARP with INO-1001 reshifted the location of the apoptotic marker to the axons in the ipsilateral striatum. Thus, PARP inhibition is neuroprotective and regulates the ischemic nuclear translocation of AIF in stroke.

The discovery and synthesis of novel adenosine substituted 2,3-dihydro-1H-isoindol-1-ones: potent inhibitors of poly(ADP-ribose) polymerase-1 (PARP-1).

A series of novel 4-(N-acyl)-2,3-dihydro-1H-isoindol-1-ones have been prepared from methyl-3-nitro-2-methylbenzoate and linked through various spacers to the adenosine derivatives 11 and 12. We found that potent inhibition of poly(ADP-ribose)polymerase-1 (PARP-1) was achieved when isoindolinone was linked to adenosine by a spacer group of a specific length. Introduction of piperazine and succinyl linkers between the isoindolinone and adenosine core structures resulted in highly potent compounds 8a and 10b, which showed IC(50) values of 45 and 100 nM, respectively.

Poly(ADP-ribose) polymerase inhibitors.

Poly(ADP-ribose) polymerase-1 (PARP-1) is the principal member of the PARP enzyme family consisting of PARP-1 and several recently identified novel poly(ADP-ribosyl)ating enzymes. PARP-1 functions as a DNA damage sensor and signalling molecule. Upon binding to DNA breaks, activated PARP cleaves NAD(+) into nicotinamide and ADP-ribose and polymerizes the latter onto nuclear acceptor proteins including histones, transcription factors and PARP itself. This Poly(ADP-ribosyl)ation contributes to inflammatory signal transduction processes. In addition, oxidative stress-induced overactivation of PARP consumes NAD(+) and consequently ATP, culminating in cell dysfunction or necrosis. Activation of PARP has been implicated in the pathogenesis of stroke, myocardial ischemia, diabetes, diabetes-associated cardiovascular dysfunction, shock, traumatic central nervous system injury, arthritis, colitis, allergic encephalomyelitis and various other forms of inflammation. Therefore, inhibition of PARP by pharmacological agents may prove useful for the therapy of these diseases, as has been shown in preclinical animal models. Moreover, PARP inhibitors may have additional, potential utility as anticancer agents, radiosensitizers and antiviral agents. In the present article we overview the structures and pharmacological actions of various pharmacological classes of compounds which inhibit the catalytic activity of PARP.

Part II: beneficial effects of the peroxynitrite decomposition catalyst FP15 in murine models of arthritis and colitis.

BACKGROUND: Peroxynitrite is a reactive oxidant species produced from nitric oxide and superoxide, which has been indirectly implicated in the pathogenesis of many inflammatory conditions including arthritis and colitis. Here, using a novel peroxynitrite decomposition catalyst, FP15, we directly investigate the role of peroxynitrite in the pathogenesis of arthritis and colitis in rodent models. METHODS: Arthritis was induced in mice by intradermal collagen injection; incidence and severity of arthritis was monitored using a macroscopic scoring system. At the end of the experiment paws were taken for determination of neutrophil infiltration (myeloperoxidase [MPO] activity), oxidative stress (malondialdehyde [MDA] level), and cytokine/chemokine levels. Colitis was induced in mice by 5% dextran sodium sulfate (DSS) in their drinking water. Colitis symptoms were assessed 10 days later, the parameters determined included body weight, rectal bleeding, colon length, colonic MPO and MDA levels, and colon histologic damage. RESULTS: Treatment with FP15 significantly reduced the inflammation and oxidative stress in arthritis and colitis. FP15 reduced both the incidence and severity of arthritis in mice and this was associated with reduced paw MPO and MDA levels. Similarly, in colitis, FP15 reduced colon damage, and this was associated with reduced colon neutrophil infiltration and oxidative stress. CONCLUSIONS: The protective effect of FP15 suggests that peroxynitrite plays a significant pathogenetic role in arthritis and colitis in the currently employed rodent models. Further work is needed to determine whether neutralization of peroxynitrite also represents a promising strategy to treat human inflammatory diseases such as arthritis and colitis.

Aerosolized linear polyethylenimine-nitric oxide/nucleophile adduct attenuates endotoxin-induced lung injury in sheep.

Pulmonary hypertension and edema are mainstays of acute lung injury (ALI). We synthesized linear polyethylenimine-nitric oxide/nucleophile adduct (DS-1), a water-soluble nitric oxide donor, and demonstrated that it is a potent relaxant of precontracted rat aortic rings without inducing desensitization. Moreover, DS-1 does not suppress the viability of human pulmonary epithelial cells in vitro. We also tested whether DS-1 counteracts ALI in endotoxemic sheep. Animals were instrumented for a chronic study. In 16 awake, spontaneously breathing sheep, Escherichia coli endotoxin (10 ng/kg/minute) was infused for 8 hours. From 2 hours of endotoxemia, sheep received either nebulized DS-1 (1 mg/kg/hour) or isotonic saline. DS-1 reduced endotoxin-induced rises in pulmonary arterial and microwedge pressures and vascular resistance index by 40-70%. In parallel, DS-1 decreased the accumulation of extravascular lung water by 60-70% and reduced the increment in right ventricle stroke work index and the falls in right ventricle ejection fraction, stroke volume, and left ventricle stroke work indices. Furthermore, DS-1 reduced venous admixture and improved arterial oxygen saturation. In four healthy animals, DS-1 alone slightly increased arterial oxygenation but had no other effects. Thus, aerosolized DS-1 attenuates endotoxin-induced ALI in sheep by reducing pulmonary hypertension and edema and improving myocardial function and gas exchange.

Novel phenanthridinone inhibitors of poly (adenosine 5'-diphosphate-ribose) synthetase: potent cytoprotective and antishock agents.

OBJECTIVE: To synthesize novel inhibitors of the nuclear enzyme poly(adenosine 5'-diphosphate [ADP]-ribose) synthetase (PARS), also known as poly(ADP-ribose) polymerase (PARP), and to test them in in vitro models of oxidant-induced cytotoxicity and in endotoxin and splanchnic occlusion-reperfusion-induced shock. DESIGN: Randomized, prospective laboratory study. SETTING: Research laboratory. SUBJECTS: Murine macrophages, thymocytes, and endothelial cells; Balb/c mice and Wistar rats. INTERVENTIONS: Macrophages and endothelial cells were treated with peroxynitrite and bleomycin to induce PARS activation, and thymocytes were treated with peroxynitrite to induce cell necrosis. Novel PARS inhibitors were synthesized and used to reduce PARS activation and to reverse cytotoxicity. Balb/c mice were subjected to splanchnic occlusion and reperfusion and were pretreated with various doses (1-10 mg/kg intraperitoneally) of PJ34, a selected, potent, water-soluble PARS inhibitor. The passage of fluorescein isothiocyanate-conjugated dextran (4 kDa) was analyzed in everted gut ileal sacs incubated ex vivo as an index of gut permeability. Wistar rats were subjected to Escherichia coli bacterial lipopolysaccharide (40 mg/kg intraperitoneally). PJ34 was also used at 10 mg/kg intraperitoneally, 1 hr before lipopolysaccharide or at 25 mg/kg intraperitoneally 1 hr after lipopolysaccharide treatment. Serum concentrations of indicators or multiple organ injury, concentrations of various proinflammatory mediators, and tissue concentrations of myeloperoxidase and malondialdehyde were measured. In addition, survival rates and vascular contractile and relaxant responses were recorded. MEASUREMENTS AND MAIN RESULTS: Appropriate modifications of the phenanthridinone core structure yielded significant increases in the potency of the compounds, both as PARS inhibitors and as cytoprotective agents. The compound N-(6-oxo-5,6-dihydro-phenanthridin-2-yl) -N,N-dimethylacetamide (designated as PJ34) was one of the potent PARS inhibitors of the series, and it dose-dependently protected against thymocyte necrosis, with a half-maximal restoration of cell viability of 35 nM and complete protection at 200 nM. PARS activation also was visualized by immunohistochemistry and was dose-dependently suppressed by PJ34. The effect of PJ34 was dose-dependently reversed by excess nicotinamide adenine dinucleotide (oxidized). The PARS inhibitors dose-dependently suppressed proinflammatory cytokine and chemokine production and restored viability in immunostimulated macrophages. PJ34 was selected for the subsequent in vivo studies. PJ34 significantly protected against splanchnic reperfusion-induced intestinal hyperpermeability in the mouse. PJ34 reduced peak plasma concentrations of tumor necrosis factor-alpha, interleukin-1beta, and nitrite/nitrate in the plasma of lipopolysaccharide-treated rats. PJ34 ameliorated the lipopolysaccharide-induced increases in indexes of liver and kidney failure and concentrations of myeloperoxidase and malondialdehyde in the lung and gut. Lipopolysaccharide elicited vascular dysfunction, which was normalized by PJ34. Lipopolysaccharide-induced mortality was reduced by PJ34 (both pre- and posttreatment). CONCLUSIONS: The novel series of phenanthridinone PARS inhibitors have potent cytoprotective effects in vitro and significant protective effects in shock and reperfusion injury in rodent models in vivo.

Poly(ADP-Ribose) polymerase inhibition reduces reperfusion injury after heart transplantation.

The aim of the present study was to investigate the effects of the novel poly(ADP-ribose) polymerase (PARP) inhibitor PJ34 (N-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide) on myocardial and endothelial function after hypothermic ischemia and reperfusion in a heterotopic rat heart transplantation model. After a 1-hour ischemic preservation, reperfusion was started either after application of placebo or PJ34 (3 mg/kg). The assessment of left ventricular pressure-volume relations, total coronary blood flow, endothelial function, myocardial high energy phosphates, and histological analysis were performed at 1 and 24 hours of reperfusion. After 1 hour, myocardial contractility and relaxation, coronary blood flow, and endothelial function were significantly improved and myocardial high energy phosphate content was preserved in the PJ34-treated animals. Improved transplant function was also seen with treatment with another, structurally different PARP inhibitor, 5-aminoisoquinoline. The PARP inhibitors did not affect baseline cardiac function. Immunohistological staining confirmed that PJ34 prevented the activation of PARP in the transplanted hearts. The activation of P-selectin and ICAM-1 was significantly elevated in the vehicle-treated heart transplantation group. Thus, pharmacological PARP inhibition reduces reperfusion injury after heart transplantation due to prevention of energy depletion and downregulation of adhesion molecules and exerts a beneficial effect against reperfusion-induced graft coronary endothelial dysfunction.

Mitogen-activated protein kinase phosphorylation in kidneys of beta(s) sickle cell mice.

Previous studies in beta(s) sickle cell mice demonstrated renal immunostaining for nitrotyrosine, which is putative evidence of peroxynitrite (ONOO(-)) formation. ONOO(-) is known to nitrate tyrosine residues of various enzymes, thereby interfering with phosphorylation and inactivating them. The present study examined the state of phosphorylation of mitogen-activated protein (MAP) kinase signal transduction enzymes, i.e., p38, c-Jun NH(2)-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK). Western blot performed with antibodies directed against specific phosphorylated threonine/tyrosine residues of these enzymes demonstrated reduced phosphorylation of renal p38 and a trend toward reduced phosphorylation of ERK. In contrast, phosphorylation of renal JNK was markedly increased compared with normal mice. The abundance of MAP kinase phosphatase-1 (MKP-1), a key upstream enzyme that modulates phosphorylation of MAP kinases, was not different in beta(s) versus normal mice. To determine whether nitration of tyrosine by ONOO(-) was responsible for reduced phosphorylation of p38 and ERK, mercaptoethylguanidine (MEG), a compound known to reduce inducible isoform of nitric oxide synthase activity and to scavenge ONOO(-), was administered to beta(s) mice for 5 d. MEG was found to restore phosphorylation of p38 and ERK toward normal levels. These observations provide evidence that ONOO(-) (or closely related reaction products of NO) contributes to dephosphorylation of p38 and ERK, and presumably reduces activity of these enzymes. The increased phosphorylation of JNK, which suggests activation of this signaling pathway by extracellular stress signals, may play a role in apoptosis in the kidneys of these mice. The changes in phosphorylation of MAP kinase pathways found in this study could have important consequences for regulation of nuclear transcription factors, and thus renal function and pathology in sickle cell kidneys.