Antihypertensive effect of mitochondria-targeted proxyl nitroxides

Abstract

Superoxide (O2-•) has been implicated in the pathogenesis of many human diseases including hypertension. Mitochondria-targeted superoxide scavenger mitoTEMPO reduces blood pressure; however, the structure–functional relationships in antihypertensive effect of mitochondria-targeted nitroxides remain unclear. The nitroxides are known to undergo bioreduction into hydroxylamine derivatives which reacts with O2-• with much lower rate. The nitroxides of pyrrolidine series (proxyls) are much more resistant to bioreduction compared to TEMPOL derivatives suggesting that mitochondria-targeted proxyls can be effective antioxidants with antihypertensive activity. In this work we have designed and studied two new pyrrolidine mitochondria targeted nitroxides: 3-[2-(triphenyphosphonio)acetamido]- and 3-[2-(triphenyphosphonio) acetamidomethyl]-2,2,5,5-tetramethylpyrrolidine-1-oxyl (mCP2) and (mCP1). These new mitochondria targeted nitroxides have 3- to 7-fold lower rate constants of the reaction with O2-• compared with mitoTEMPO; however, the cellular bioreduction of mCP1 and mCP2 was 3- and 2-fold slower. As a consequence incubation with cells afforded much higher intracellular concentration of mCP1 and mCP2 nitroxides compared to mitoTEMPO nitroxide. This has compensated for the difference in the rate of O2-• scavenging and all nitroxides similarly protected mitochondrial respiration in H2O2 treated endothelial cells. Treatment of hypertensive mice with mCP1 and mCP2 (1.4 mg/kg/day) after onset of angiotensin II-induced hypertension significantly reduced blood pressure to 133±5 mmHg and 129±6 mmHg compared to 163±5 mmHg in mice infused with angiotensin II alone. mCP1 and mCP2 reduced vascular O2-• and prevented decrease of endothelial nitric oxide production. These data indicate that resistance to bioreduction play significant role in antioxidant activity of nitroxides. Studies of nitroxide analogs such as mCP1 and mCP2 may help in optimization of chemical structure of mitochondria-targeted nitroxides for improved efficacy and pharmacokinetics of these drugs in treatment of hypertension and many other conditions including atherosclerosis, diabetes and degenerative neurological disorders in which mitochondrial oxidative stress seems to play a role.

Cellular accumulation and antioxidant activity of acetoxymethoxycarbonyl pyrrolidine nitroxides

“…We have previously shown that mitochondria-targeted superoxide dismutase mimetic mitoTEMPO reduces mitochondrial superoxide in endothelial cells treated with hormone angiotensin II or H 2 O 2 while untargeted analogue TEMPOL was not effective [10,19]. In this work, we have compared the antioxidant effect of mitoTEMPO and DCP-AM2 on mitochondrial superoxide level.…”

“…In contrast, incubation of endothelial cells with DCP did not lead to significant consumption of DCP nitroxide from the buffer and only minor accumulation of DCP in cells was observed (Figure 2). Interestingly, incubation of cells with mitoTEMPO lead to significant absorption of nitroxide from the buffer; however, no significant EPR signal of mitoTEMPO in cells was found due to rapid cellular bioreduction [10]. …”

“…To a stirred solution of 3-chloro acetamido-Proxyl [10] (0.483 g, 3 mmol) in acetone (10 ml), potassium carbonate (0.828 g, 6 mmol) and iminodiacetic acid dimethyl ester (0.700 g, 3 mmol) were added. After 6 h of stirring at room temperature, the reaction mixture was diluted with water (20 ml) and extracted with CHCl 3 (2 × 15 ml).…”

“…Intracellular accumulation retards clearance and can strongly increase the biological activity of nitroxides. Recently, we have described the antihypertensive activity of mitochondria-targeted piperidine and pyrrolidine nitroxides with triphenylphosphonium cationic groups [10] which are actively accumulated in cells and in mitochondria due to transmembrane electrostatic potential. Another approach implies the introduction of acetoxymethoxycarbonyl groups [11,12] which provide high cellular retention [13], very good tissue permeability and sustain EPR signal in vivo [14].…”

Mitochondrial Dysfunction and Redox Imbalance as a Diagnostic Marker of “Free Radical Diseases”

“…Many researchers have reported that stable nitroxide radicals are appropriate redox sensors for ROS imaging and undergo bioreduction to hydroxylamine derivatives, which can react with superoxide (85)(86)(87). For example, nitroxides as mito-TEMPO and mito-carboxy-PROXYL are mitochondria-targeted derivatives of TEMPOL and carboxy-PROXYL, which currently are widely used as redox-sensors for detection of abnormal superoxide generation in living cells and tissues (2,28,37,(88)(89)(90)(91).…”