Clinical pharmacology and tolerability of REC-994, a redox-cycling nitroxide compound, in randomized phase 1 dose-finding studies.

Cerebral cavernous malformation (CCM) has variable clinical symptoms, including potentially fatal hemorrhagic stroke. Treatment options are very limited, presenting a large unmet need. REC-994 (also known as tempol), identified as a potential treatment through an unbiased drug discovery platform, is hypothesized to treat CCMs through a reduction in superoxide, a reactive oxygen species. We investigated the safety, tolerability, and pharmacokinetic profile of REC-994 in healthy volunteers. Single- and multiple-ascending dose (SAD and MAD, respectively) studies were conducted in adult volunteers (ages 18-55). SAD study participants received an oral dose of REC-994 or placebo. MAD study participants were randomized 3:1 to oral doses of REC-994 or matching placebo, once daily for 10 days. Thirty-two healthy volunteers participated in the SAD study and 52 in the MAD study. Systemic exposure increased in proportion to REC-994 dose after single doses of 50-800 mg and after 10 days of dosing over the 16-fold dose range of 50-800 mg. Median Tmax and mean t1/2 were independent of dose in both studies, and the solution formulation was more rapidly absorbed. REC-994 was well tolerated. Treatment-emergent adverse effects across both studies were mild and transient and resolved by the end of the study. REC-994 has a favorable safety profile and was well tolerated in single and multiple doses up to 800 mg with no dose-limiting adverse effects identified. Data support conducting a phase 2 clinical trial in patients with symptomatic CCM.

Targeting transcription of MCL-1 sensitizes HER2-amplified breast cancers to HER2 inhibitors.

Human epidermal growth factor receptor 2 gene (HER2) is focally amplified in approximately 20% of breast cancers. HER2 inhibitors alone are not effective, and sensitizing agents will be necessary to move away from a reliance on heavily toxic chemotherapeutics. We recently demonstrated that the efficacy of HER2 inhibitors is mitigated by uniformly low levels of the myeloid cell leukemia 1 (MCL-1) endogenous inhibitor, NOXA. Emerging clinical data have demonstrated that clinically advanced cyclin-dependent kinase (CDK) inhibitors are effective MCL-1 inhibitors in patients, and, importantly, well tolerated. We, therefore, tested whether the CDK inhibitor, dinaciclib, could block MCL-1 in preclinical HER2-amplified breast cancer models and therefore sensitize these cancers to dual HER2/EGFR inhibitors neratinib and lapatinib, as well as to the novel selective HER2 inhibitor tucatinib. Indeed, we found dinaciclib suppresses MCL-1 RNA and is highly effective at sensitizing HER2 inhibitors both in vitro and in vivo. This combination was tolerable in vivo. Mechanistically, liberating the effector BCL-2 protein, BAK, from MCL-1 results in robust apoptosis. Thus, clinically advanced CDK inhibitors may effectively combine with HER2 inhibitors and present a chemotherapy-free therapeutic strategy in HER2-amplified breast cancer, which can be tested immediately in the clinic.

A Phase I Study of Dinaciclib in Combination With MK-2206 in Patients With Advanced Pancreatic Cancer.

The combination of drugs targeting Ral and PI3K/AKT signaling has antitumor efficacy in preclinical models of pancreatic cancer. We combined dinaciclib (small molecule cyclin dependent kinase inhibitor with MK-2206 (Akt inhibitor) in patients with previously treated/metastatic pancreatic cancer. Patients were treated with dinaciclib (6-12 mg/m2 i.v.) and MK-2206 (60-135 mg p.o.) weekly. Tumor biopsies were performed to measure pAKT, pERK, and Ki67 at baseline and after one completed cycle (dose level 2 and beyond). Thirty-nine patients participated in the study. The maximum tolerated doses were dinaciclib 9 mg/m2 and MK-2206 135 mg. Treatment-related grade 3 and 4 toxicities included neutropenia, lymphopenia, anemia, hyperglycemia, hyponatremia, and leukopenia. No objectives responses were observed. Four patients (10%) had stable disease as their best response. At the recommended dose, median survival was 2.2 months. Survival rates at 6 and 12 months were 11% and 5%, respectively. There was a nonsignificant reduction in pAKT composite scores between pretreatment and post-treatment biopsies (mean 0.76 vs. 0.63; P = 0.635). The combination of dinaciclib and MK-2206 was a safe regimen in patients with metastatic pancreatic cancer, although without clinical benefit, possibly due to not attaining biologically effective doses. Given the strong preclinical evidence of Ral and AKT inhibition, further studies with better tolerated agents should be considered.

Mechanistic study of mtROS-JNK-SOD2 signaling in bupivacaine-induced neuron oxidative stress.

Manganese superoxide dismutase (SOD2) is a key enzyme to scavenge free radical superoxide in the mitochondrion. SOD2 deficiency leads to oxidative injury in cells. Bupivacaine, a local anesthetic commonly used in clinic, could induce neurotoxic injury via oxidative stress. The role and the mechanism of SOD2 regulation in bupivacaine-induced oxidative stress remains unclear. Here, bupivacaine was used to treat Sprague-Dawley rats with intrathecal injection and culture human neuroblastoma cells for developing vivo injury model and vitro injury model. The results showed that bupivacaine caused the over-production of mitochondrial reactive oxygen species (mtROS), the activation of C-Jun N-terminal kinase (JNK), and the elevation of SOD2 transcription. Decrease of mtROS with N-acetyl-L-cysteine attenuated the activation of JNK and the increase of SOD2 transcription. Inhibition of JNK signaling with a small interfering RNA (siRNA) or with sp600125 down-regulated the increase of SOD2 transcription. SOD2 gene knock-down exacerbated bupivacaine-induced mtROS generation and neurotoxic injury but had no effect on JNK phosphorylation. Mito-TEMPO (a mitochondria-targeted antioxidant) could protect neuron against bupivacaine-induced toxic injury. Collectively, our results confirm that mtROS stimulates the transcription of SOD2 via activating JNK signaling in bupivacaine-induced oxidative stress. Enhancing antioxidant ability of SOD2 might be crucial in combating bupivacaine-induced neurotoxic injury.

Effect of reactive oxygen species of the psoas major muscle in complete Freund's adjuvant-induced inflammatory pain in rats.

Lower limb pain is a common clinical disease that affects millions of people worldwide. It is found in previous studies that reactive oxygen species is closely related to neuropathic, cancer, chemotherapy, and inflammatory pain, which can be relieved by reactive oxygen species scavengers. Furthermore, acupuncture or electroacupuncture on the psoas major muscle has a great effect on adjuvant-induced arthritis and lower back pain. In our study, we investigated the function of reactive oxygen species scavengers locally injecting into the ipsilateral psoas major muscle on complete Freund's adjuvant-induced inflammatory pain. Our results demonstrated that in the development of complete Freund's adjuvant-induced inflammatory pain, early local continuous application of N-tert-Butyl-α-phenylnitrone (PBN, 1 and 5 mg/kg/0.2 ml) on the ipsilateral psoas major muscle effectively reduced mechanical and cold hyperalgesia. However, intraperitoneal injection of PBN (1 and 5 mg/kg) or local injection of PBN (1 and 5 mg/kg/0.2 ml) into contralateral psoas major muscle, ipsilateral quadratus lumborum, and ipsilateral erector spinae showed limited effect. In the developed inflammatory pain model, local injection of PBN into the ipsilateral psoas major muscle also alleviated pain and paw edema. In addition, reactive oxygen species level increased in ipsilateral psoas major muscle at seven days after complete Freund's adjuvant injection. In general, PBN reduces complete Freund's adjuvant-evoked inflammatory pain by inhibiting reactive oxygen species in the psoas major muscle.

Novel neuroprotection using antioxidant nanoparticles in a mouse model of head trauma.

INTRODUCTION: Free radicals and reactive oxygen species are related to deteriorating pathological conditions after head trauma because of their secondary effects. 2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO) scavenges free radicals; however, this molecule is also toxic. Here, we have evaluated the neuroprotective effect of antioxidant nanoparticles, which consisted of a novel core-shell type nanoparticle containing 4-amino-TEMPO, that is, redox-active nitroxide radical-containing nanoparticles (RNPs). METHODS: Institute of Cancer Research mice were subjected to a head-impact procedure, randomly divided into four groups and intravenously (3 mg/kg) administered phosphate-buffered saline, TEMPO, micelle (a self-assembling block copolymer micelle without a TEMPO moiety), or RNP through the tail vein immediately thereafter and intraperitoneally at days 1, 3, and 5 after traumatic brain injury (TBI). The RNP distribution was detected by rhodamine labeling. Cognitive behavior was assessed using the neurological severity score and a rotarod test at days 1, 3, and 7 following TBI, and contusion volume was measured at day 7 after TBI. Free radical-scavenging capacity was analyzed by electron paramagnetic resonance on day 1 after TBI, and immunostaining was used to observe mobilization of microglia (Iba-1) and rescued neuronal cells (NeuN). RESULTS: Redox-active nitroxide radical-containing nanoparticle was detected in the microvessels around the injured area in the brain. Cognitive behavior assessment was significantly better, and contusion volume was significantly smaller in the RNP group compared with the other groups. Superoxide anion scavenging capacity was significantly higher in the RNP group, and neuronal loss was significantly suppressed around the injured area at day 7 after TBI. Furthermore, in the RNP group, neurodegenerative microglia production was suppressed at days 3 and 7 after TBI, whereas neuroprotective microglia production was higher at day 7 after TBI. CONCLUSION: The RNP administration after TBI improved cognitive behavior and reduced contusion volume by improving reactive oxygen species scavenging capacity. Therefore, RNP may have a neuroprotective effect after TBI. LEVEL OF EVIDENCE: Therapeutic test.

Hernia Repair Strength Enhanced With Antioxidants.

BACKGROUND: Incisional hernia is one of the most common complications of abdominal surgery, and repairs are associated with significant recurrence rates. Mesh repairs are associated with the best outcomes, but failures are not uncommon. Doxycycline has been demonstrated to enhance mesh hernia repair outcomes with associated increases in collagen deposition and improved tensiometric strength. This study compares the outcomes of incisional hernia repair with doxycycline administration and the antioxidant tempol. MATERIALS AND METHODS: Twenty-eight male Sprague Dawley rats underwent a midline hernia creation and an intraabdominal polypropylene mesh repair. The animals were administered saline, doxycycline, tempol, or both, daily for 8 wk. The abdominal wall was harvested at 8 wk and tensiometric strength and biochemical analysis was performed. RESULTS: The tensiometric strength of the repair was increased in all experimental groups. Collagen type 1 deposition was increased, and collagen type 3 deposition was decreased in each of the experimental groups relative to control. There was no difference in MMP-2 and MMP-9 levels between control and experimental groups. CONCLUSIONS: The hernia repair strength is equally enhanced with the administration of doxycycline or tempol. Dual therapy provided no benefit over treatment with either single agent. All treatment groups had an increase in collagen type 1:3 ratios, but the mechanism is not well understood. The benefits of antioxidant treatment following hernia repair are similar to treatment with doxycycline. Given the high frequency of incisional hernia repair failures, this study has implications for improving outcomes following ventral hernia repair through the use of either doxycycline or antioxidant therapy.

Effect of Tempol, a Membrane-Permeable Free Radical Scavenger, on In Vitro Model of Eye Inflammation on Rabbit Corneal Cells.

Purpose: Inflammatory corneal diseases such as bacterial keratitis provoke severe injury to the visual functions and physical structure, leading to opaqueness, wounding, damage to the cornea, and even long-lasting vision loss. Usually antioxidant substances have been of great attention as candidate therapies in the management of keratitis in both humans and animals. Based on the findings, the aim of our research was to examine the effects of Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl), a membrane-permeable free radical scavenger with exclusive antioxidant properties, on in vitro model of eye inflammation of rabbit corneal cells stimulated with lipopolysaccharide (LPS) (Seruminstitute Rabbit Cornea). Methods: The cells were pretreated with Tempol and incubated with LPS for 24 h. LPS stimulation triggered increased cellular mortality, oxidative stress, cytokine levels expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß and IL-6, and also enhanced prostaglandin E2 (PGE2) levels and cyclooxygenase-2 (COX-2) expression. Results: Pretreatment with Tempol (3 mM) significantly increased cell viability and antioxidant activity as well as decreased reactive oxygen species production, cytokines, PGE2 levels, and COX-2 expression. Conclusions: Taken together, Tempol could be a new therapeutic strategy for management of ocular inflammatory disorders for clinical and veterinary use.

A self-assembled, ROS-responsive Janus-prodrug for targeted therapy of inflammatory bowel disease.

A self-assembled and oxidation-degradable Janus-prodrug, termed as Bud-ATK-Tem (B-ATK-T), was fabricated by ROS-responsive aromatized thioketal (ATK) linked anti-inflammatory drug budesonide (Bud) and antioxidant tempol (Tem). Benefiting from the hydrophobic interactions and π-π stacking interactions of ATK, prodrug B-ATK-T could self-assemble into nanoparticles (NP) in water containing lecithin and DSPE-PEG2K. The morphology of B-ATK-T NP (approximate 100-120nm) was confirmed to be regular spherical by transmission electron microscope. B-ATK-T NP was endowed high drug loading content with 41.23% for Bud and 15.55% for Tem. The rapid drug release from B-ATK-T NP proceeded in an extensive reactive oxygen species (ROS)-dependent manner. More than 98% of Bud and Tem in B-ATK-T NP could release in the mimic inflammation microenvironment or phorbol-12-myristate-13-acetate (PMA)-stimulated macrophages within short time. The release of drugs in a simultaneous and proportional manner ensures that B-ATK-T NP can increase the combined efficacy of anti-inflammation and anti-oxidation. It is worth noting that B-ATK-T NP could be passively accumulated and dramatically increasing the maximum drugs concentration in the inflamed colon of mice with inflammatory bowel disease (IBD) by oral route, and avoiding potential systemic side effects. B-ATK-T NP could not only relieve colitis via inhibiting the expression of oxidative and proinflammatory mediators more than combination of free drugs, but also significantly reduce colitis-caused death. Taken together, the self-assembled, Janus-prodrug B-ATK-T NP is a promising candidate therapies for IBD, even for other inflammatory diseases.

Hsp22 overexpression induces myocardial hypertrophy, senescence and reduced life span through enhanced oxidative stress.

H11 kinase/Hsp22 (Hsp22) is a small heat shock protein, which, when overexpressed cardiac specifically in transgenic (TG) mice, induces stable left ventricular (LV) hypertrophy. Hsp22 also increases oxidative phosphorylation and mitochondrial reactive oxygen species (ROS) production, mechanisms mediating LV hypertrophy, senescence and reduced lifespan. Therefore, we investigated whether ROS production mediates LV hypertrophy, senescence and reduced life span in Hsp22 TG mice. Survival curves revealed that TG mice had a 48% reduction in their mean life span compared to wild type (WT) mice. This was associated with a significant increase in senescence markers, such as p16, p19 mRNA levels as well as the percentage of ß-galactosidase positive cells and telomerase activity. Oxidized (GSSG)/reduced (GSH) glutathione ratio, an indicator of oxidative stress, and ROS production from 3 major cellular sources was measured in cardiac tissue. Hearts from TG mice exhibited a decrease in GSH/GSSG ratio together with increased ROS production from all sources. To study the role of ROS, mice were treated with the antioxidant Tempol from weaning to their sacrifice. Chronic Tempol treatment abolished oxidative stress and overproduction of ROS, and reduced myocardial hypertrophy and Akt phosphorylation in TG mice. Tempol also significantly extended life span and prevented aging markers in TG mice. Taken together these results show that overexpression of Hsp22 increases oxidative stress responsible for the induction of hypertrophy and senescence and ultimately reduction in life span.

Tempol, a superoxide dismutase mimetic agent, reduces cisplatin-induced nephrotoxicity in rats.

Cisplatin (CP) is one of the most potent anti-cancer drugs used against different types of cancer. Its use is limited due to its nephrotoxicity. This study is aimed to evaluate the role of a super oxide dismutase (SOD) mimetic agent, tempol, in protection against CP nephrotoxicity in rats. Animals were divided into four groups: Group-1: Normal control group, Group-2: CP group (single dose of CP 6 mg/kg, i.p.), Group-3 and Group-4: Tempol-treated groups (50 mg/kg p.o. and 100 mg/kg p.o. respectively) daily for a week before CP injection and continued for an additional four days after CP injection. Urine and blood samples were collected for the evaluation of kidney function including serum creatinine, BUN, cystatin-c, and creatinine clearance. In addition, western blotting was used to determine urine lipocalin-2 content. Furthermore, kidney tissue was collected for the determination of oxidative stress markers, caspase-3 expression, and histopathological examination. We noticed that both doses of tempol significantly improved kidney function, which was deteriorated by CP injection. Tempol significantly elevated kidney glutathione (GSH) content and SOD activity, and decreased kidney lipid peroxidation and NOx production. Tempol also significantly decreased kidney caspase-3 expression which was elevated by CP toxicity. Thus, we conclude that tempol can protect against CP nephrotoxicity. We noticed that both doses of tempol are effective in ameliorating CP-nephrotoxicity.

Effects of Antioxidant Tempol on Systematic Inflammation and Endothelial Apoptosis in Emphysematous Rats Exposed to Intermittent Hypoxia.

PURPOSE: Obstructive sleep apnea and chronic obstructive pulmonary disease are independent risk factors of cardiovascular disease (CVD), and their coexistence is known as overlap syndrome (OS). Endothelial dysfunction is the initial stage of CVD; however, underlying mechanisms linking OS and CVD are not well understood. The aim of this study was to explore whether OS can lead to more severe inflammation and endothelial apoptosis by promoting endothelial dysfunction, and to assess the intervention effects of antioxidant tempol. MATERIALS AND METHODS: Male Wistar rats (n=66) were exposed to normal oxygen [normal control (NC) group], intermittent hypoxia (IH group), cigarette smoke (CH group), as well as cigarette smoke and IH (OS group). Tempol intervention was assessed in OS group treated with tempol (OST group) or NaCl (OSN group). After an 8-week challenge, lung tissues, serum, and fresh blood were harvested for analysis of endothelial markers and apoptosis. RESULTS: The levels of intracellular adhesion molecule-1, vascular cellular adhesion molecule-1, and apoptosis in circulating epithelial cells were the highest in OS group and the lowest in NC group. These levels were all greater in IH group than in CH group, and were lower in OST group than in OS and OSN groups (all p<0.001). CONCLUSION: Synergistic effects of IH with cigarette smoke-induced emphysema produce a greater inflammatory status and endothelial apoptosis. OS-related inflammation and endothelial cell apoptosis may play important roles in promoting cardiovascular dysfunction, and antioxidant tempol could achieve a partial protective effect.

Angiotensin II, a stress-related neuropeptide in the CNS, facilitates micturition reflex in rats.

BACKGROUND AND PURPOSE: We investigated the effects of centrally administered stress-related neuropeptide, angiotensin II, on the micturition reflex and the downstream signalling pathways in rats. EXPERIMENTAL APPROACH: Male Wistar rats were anaesthetized with urethane for cystometry before and after i.c.v. administration of vehicle or angiotensin II (30 pmol). Muscimol (a GABAA receptor agonist) or baclofen (a GABAB receptor agonist) was i.c.v. administered 30 min before or 15 min after central angiotensin II administration. Telmisartan [an angiotensin II type 1 (AT1 ) receptor antagonist], valsartan (an AT1 receptor antagonist), PD123319 (an AT2 receptor antagonist), U-73122 (a PLC inhibitor), chelerythrine chloride (a PKC inhibitor), apocynin (a NADPH oxidase inhibitor) or tempol (an antioxidant) was centrally administered 30 min before central angiotensin II administration. KEY RESULTS: Centrally administered angiotensin II significantly shortened the intercontraction interval and decreased the voided volume and bladder capacity without altering the maximum voiding pressure, post-voiding residual urine volume or voiding efficacy. Muscimol, baclofen, telmisartan, valsartan, U-73122, chelerythrine chloride, apocynin or tempol pretreatment significantly suppressed the reduction in intercontraction interval induced by central angiotensin II. Post-treatment with muscimol or baclofen also ameliorated the decrease in intercontraction interval induced by central angiotensin II. CONCLUSIONS AND IMPLICATIONS: Angiotensin II in the CNS facilitates micturition reflex by inhibiting central GABAergic activity and activating the AT1 receptor/PLC/PKC/NADPH oxidase/superoxide anion pathway.

Endothelinergic Contractile Hyperreactivity in Rat Contralateral Carotid to Balloon Injury: Integrated Role for ETB Receptors and Superoxide Anion.

Temporal consequences of neurocompensation to balloon injury on endothelinergic functionality in rat contralateral carotid were evaluated. Rats underwent balloon injury in left carotid and were treated with CP-96345 (NK1 antagonist). Concentration-response curves for endothelin-1 were obtained in contralateral (right) carotid at 2, 8, 16, 30, or 45 days after surgery in the absence or presence of BQ-123 (ETA antagonist), BQ-788 (ETB antagonist), or Tempol (superoxide-dismutase mimic). Endothelin-1-induced calcium mobilization was evaluated in functional assays carried out with BQ-123, BQ-788, or Tempol. Endothelin-1-induced NADPH oxidase-driven superoxide generation was measured by lucigenin chemiluminescence assays performed with BQ-123 or BQ-788. Endothelin-1-induced contraction was increased in contralateral carotid from the sixteenth day after surgery. This response was restored in CP-96345-treated rats. Endothelium removal or BQ-123 did not change endothelin-1-induced contraction in contralateral carotid. This response was restored by BQ-788 or Tempol. Contralateral carotid exhibited an increased endothelin-1-induced calcium mobilization, which was restored by BQ-788 or Tempol. Contralateral carotid exhibited an increased endothelin-1-induced lucigenin chemiluminescence, which was restored by BQ-788. We conclude that the NK1-mediated neurocompensatory response to balloon injury elicits a contractile hyperreactivity to endothelin-1 in rat contralateral carotid by enhancing the muscular ETB-mediated NADPH oxidase-driven generation of superoxide, which activates calcium channels.

Indole-TEMPO conjugates alleviate ischemia-reperfusion injury via attenuation of oxidative stress and preservation of mitochondrial function.

Mitochondrial oxidative damage contributes to a wide range of pathologies including ischemia/reperfusion injury. Accordingly, protecting mitochondria from oxidative damage should possess therapeutic relevance. In the present study, we have designed and synthesized a series of novel indole-TEMPO conjugates that manifested good anti-inflammatory properties in a murine model of xylene-induced ear edema. We have demonstrated that these compounds can protect cells from simulated ischemia/reperfusion (s-I/R)-induced reactive oxygen species (ROS) overproduction and mitochondrial dysfunction. Furthermore, we have demonstrated that indole-TEMPO conjugates can attenuate organ damage induced in rodents via intestinal I/R injury. We therefore propose that the pharmacological profile and mechanism of action of these indole-TEMPO conjugates involve convergent roles, including the ability to decrease free radical production via lipid peroxidation which couples to an associated decrease in ROS-mediated activation of the inflammatory process. We further hypothesize that the protective effects of indole-TEMPO conjugates partially reside in maintaining optimal mitochondrial function.

Non-invasive imaging of the levels and effects of glutathione on the redox status of mouse brain using electron paramagnetic resonance imaging.

Glutathione (GSH) is the most abundant non-protein thiol that buffers reactive oxygen species in the brain. GSH does not reduce nitroxides directly, but in the presence of ascorbates, addition of GSH increases ascorbate-induced reduction of nitroxides. In this study, we used electron paramagnetic resonance (EPR) imaging and the nitroxide imaging probe, 3-methoxycarbonyl-2,2,5,5-tetramethyl-piperidine-1-oxyl (MCP), to non-invasively obtain spatially resolved redox data from mouse brains depleted of GSH with diethyl maleate compared to control. Based on the pharmacokinetics of the reduction reaction of MCP in the mouse heads, the pixel-based rate constant of its reduction reaction was calculated as an index of the redox status in vivo and mapped as a "redox map". The obtained redox maps from control and GSH-depleted mouse brains showed a clear change in the brain redox status, which was due to the decreased levels of GSH in brains as measured by a biochemical assay. We observed a linear relationship between the reduction rate constant of MCP and the level of GSH for both control and GSH-depleted mouse brains. Using this relationship, the GSH level in the brain can be estimated from the redox map obtained with EPR imaging.

Effects of Single and Combined Losartan and Tempol Treatments on Oxidative Stress, Kidney Structure and Function in Spontaneously Hypertensive Rats with Early Course of Proteinuric Nephropathy.

Oxidative stress has been widely implicated in both hypertension and chronic kidney disease (CKD). Hypertension is a major risk factor for CKD progression. In the present study we have investigated the effects of chronic single tempol (membrane-permeable radical scavenger) or losartan (angiotensin II type 1 receptor blocker) treatment, and their combination on systemic oxidative status (plasma thiobarbituric acid-reactive substances (pTBARS) production, plasma antioxidant capacity (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid, pABTS), erythrocyte antioxidant enzymes activities) and kidney oxidative stress (kTBARS, kABTS, kidney antioxidant enzymes activities), kidney function and structure in spontaneously hypertensive rats (SHR) with the early course of adriamycin-induced nephropathy. Adult SHR were divided into five groups. The control group received vehicle, while the other groups received adriamycin (2 mg/kg, i.v.) twice in a 21-day interval, followed by vehicle, losartan (L,10 mg/kg/day), tempol (T,100 mg/kg/day) or combined T+L treatment (by gavage) during a six-week period. Adriamycin significantly increased proteinuria, plasma lipid peroxidation, kidney protein oxidation, nitrite excretion, matrix metalloproteinase-1 (MMP-1) protein expression and nestin immunostaining in the kidney. Also, it decreased kidney antioxidant defense, kidney NADPH oxidase 4 (kNox4) protein expression and abolished anti-inflammatory response due to significant reduction of kidney NADPH oxidase 2 (kNox2) protein expression in SHR. All treatments reduced protein-to-creatinine ratio (marker of proteinuria), pTBARS production, kidney protein carbonylation, nitrite excretion, increased antioxidant capacity and restored kidney nestin expression similar to control. Both single treatments significantly improved systemic and kidney antioxidant defense, bioavailability of renal nitric oxide, reduced kMMP-1 protein expression and renal injury, thus retarded CKD progression. Losartan improved blood pressure, as well as tubular injury and restored anti-inflammatory defense by reverting kNox2 expression to the control level. Interestingly, tempol was more successful in reducing systemic oxidative stress, proteinuria, kMMP-1 and glomerulosclerosis. However, combined treatment failed to overcome the beneficial effects of single treatments in slowing down the progression of ADR-induced nephropathy in SHR.

TEMPOL increases NAD(+) and improves redox imbalance in obese mice.

Continuous energy conversion is controlled by reduction-oxidation (redox) processes. NAD(+) and NADH represent an important redox couple in energy metabolism. 4-Hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) is a redox-cycling nitroxide that promotes the scavenging of several reactive oxygen species (ROS) and is reduced to hydroxylamine by NADH. TEMPOL is also involved in NAD(+) production in the ascorbic acid-glutathione redox cycle. We utilized the chemical properties of TEMPOL to investigate the effects of antioxidants and NAD(+)/NADH modulators on the metabolic imbalance in obese mice. Increases in the NAD(+)/NADH ratio by TEMPOL ameliorated the metabolic imbalance when combined with a dietary intervention, changing from a high-fat diet to a normal diet. Plasma levels of the superoxide marker dihydroethidium were higher in mice receiving the dietary intervention compared with a control diet, but were normalized with TEMPOL consumption. These findings provide novel insights into redox regulation in obesity.

The presence of serotonin in cigarette smoke - a possible mechanistic link to 5-HT-induced airway inflammation.

We previously reported the involvement of serotonin (5-HT) metabolism in cigarette smoke-induced oxidative stress in rat lung in vivo. Here, we report cigarette smoke as a source of serotonin (5-HT) to the airways and aim at investigating the effects of 5-HT on oxidative stress and inflammation in human bronchial epithelial cells (BEAS-2B). A 5-HT analog was identified to be present in aqueous phase cigarette smoke using the LC-MS/MS approach, which was later confirmed by a 5-HT enzyme-linked immune assay (EIA). Furthermore, exposure to 5-HT caused a time-dependent elevation of intracellular ROS level, which was blocked in the presence of apocynin (a NOX inhibitor). In support, the immunoblot analysis indicated that there was an increase in the expression of NOX2 time-dependently. 5-HT-induced elevation of IL-8 at both mRNA and protein levels was observed, which was inhibited by TEMPOL (a free radical scavenger), and inhibitors for p38 MAPK (SB203580) and ERK (U0126), in line with the time-dependent phosphorylation of p38 MAPK and ERK. In conclusion, our findings suggest that 5-HT presented in bronchial epithelium of smokers may be involved in cigarette smoke-induced oxidative stress and inflammation via activation of p38 MAPK and ERK pathway after the formation of free radicals.

Inhibition of Mitochondrial Fission Protein Reduced Mechanical Allodynia and Suppressed Spinal Mitochondrial Superoxide Induced by Perineural Human Immunodeficiency Virus gp120 in Rats.

BACKGROUND: Mitochondria play an important role in many cellular and physiologic functions. Mitochondria are dynamic organelles, and their fusion and fission regulate cellular signaling, development, and mitochondrial homeostasis. The most common complaint of human immunodeficiency virus (HIV)-sensory neuropathy is pain on the soles in patients with HIV, but the exact molecular mechanisms of HIV neuropathic pain are not clear. In the present study, we investigated the role of mitochondrial dynamin-related protein 1 (Drp1, a GTPase that mediates mitochondrial fission) in the perineural HIV coat glycoprotein gp120-induced neuropathic pain state. METHODS: Neuropathic pain was induced by the application of recombinant HIV-1 envelope protein gp120 into the sciatic nerve. Mechanical threshold was tested using von Frey filaments. The mechanical threshold response was assessed over time using the area under curves. Intrathecal administration of antisense oligodeoxynucleotide (ODN) against Drp1, mitochondrial division inhibitor-1 (mdivi-1), or phenyl-N-tert-butylnitrone (a reactive oxygen species scavenger) was given. The expression of spinal Drp1 was examined using western blots. The expression of mitochondrial superoxide in the spinal dorsal horn was examined using MitoSox imaging. RESULTS: Intrathecal administration of either antisense ODN against Drp1 or mdivi-1 decreased mechanical allodynia (a sensation of pain evoked by nonpainful stimuli) in the gp120 model. Intrathecal ODN or mdivi-1 did not change basic mechanical threshold in sham surgery rats. Intrathecal Drp1 antisense ODN decreased the spinal expression of increased Drp1 protein induced by peripheral gp120 application. Intrathecal phenyl-N-tert-butylnitrone reduced mechanical allodynia. Furthermore, both intrathecal Drp1 antisense ODN and mdivi-1 reversed the upregulation of mitochondrial superoxide in the spinal dorsal horn in the gp120 neuropathic pain state. CONCLUSIONS: These data suggest that mitochondrial division plays a substantial role in the HIV gp120-related neuropathic pain state through mitochondrial reactive oxygen species and provides evidence for a novel approach to treating chronic pain in patients with HIV.