Tempol targets reactive oxygen species (ROS) by acting as a superoxide dismutase (SOD) mimetic and a free radical scavenger. It catalyzes the dismutation of superoxide to hydrogen peroxide and oxygen, reducing oxidative stress. The compound also scavenges other ROS including hydroxyl radicals and peroxynitrite. Its antioxidant mechanism contributes to its cytoprotective effects in various models of oxidative stress.

Reduced cell damage and death was indicated by the temporally significantly attenuated H2O2-mediated reduction in mitochondrial respiration and increase in LDH release in rat PT cells. Tempol's advantageous effects are comparable to those of the Fe2+ chelator DEF. Nevertheless, there were no additional benefits against renal ischemia/reperfusion injury or oxidative stress-mediated PT cell damage/death with coadministration of DEF and Tempol [2].
In vitro, tempol exhibits SOD-mimetic activity by catalyzing the dismutation of superoxide. It also scavenges other reactive oxygen species including hydroxyl radicals and peroxynitrite. The compound's antioxidant activity is assessed by measuring its ability to inhibit ROS-induced damage in cell-free and cell-based systems. Tempol protects cells from oxidative stress-induced cytotoxicity and apoptosis.

The effects of resveratrol on heart function can be replicated using the SOD analog Tempol. Tempol is delivered daily by tube feeding. Mice treated with Met or Tmp had reduced PR and QTc intervals and increased heart rate compared with oral vehicle (VEH). These outcomes are similar to those achieved with RSV therapy. Pre- and post-treatment profiles of individual mice are depicted [1]. Tempol is a membrane-permeable free radical scavenger that reduces oxidative stress-mediated renal dysfunction and injury in rats. Tempol effectively reduced the increases in urea, creatinine, γGT, AST, NAG, and FENa caused by renal ischemia/reperfusion, indicating improvement in renal function and damage. Tempol also dramatically reduced renal MPO activity and MDA levels, indicating lower PMN infiltration and lipid peroxidation, respectively. Tempol lowers histological signs of renal injury associated with ischemia/reperfusion and resulted in a considerable reduction in nitrotyrosine and PARS staining, indicating reduced nitrosative and oxidative stress [2].
In vivo, tempol has been studied for its antioxidant, anti-inflammatory, and radioprotective effects in various animal models. It reduces oxidative stress in models of ischemia-reperfusion injury, hypertension, and radiation exposure. The compound also lowers blood pressure in hypertensive animal models by scavenging superoxide and improving nitric oxide bioavailability. Its cytoprotective effects are mediated through reduction of oxidative damage.

In vitro enzyme assays for tempol measure its SOD-mimetic activity. The compound's ability to dismutate superoxide is assessed using the cytochrome c reduction assay or nitro blue tetrazolium (NBT) assay. The rate of superoxide dismutation is measured spectrophotometrically, and the catalytic activity is calculated. The compound's ability to scavenge other ROS is assessed using appropriate probes.

In vitro cell-based assays for tempol assess its cytoprotective effects against oxidative stress. Cells are treated with tempol and then exposed to oxidative stress-inducing agents such as hydrogen peroxide, paraquat, or radiation. Cell viability is measured using MTT or other assays. ROS levels are measured using fluorescent probes. The compound's effects on antioxidant enzyme expression and activity are also evaluated.

In vivo animal models for tempol include models of ischemia-reperfusion injury, hypertension, radiation injury, and inflammation. The compound is administered orally or intraperitoneally, and its effects on oxidative stress markers, tissue damage, and functional outcomes are evaluated. Blood pressure is measured in hypertensive models. Pharmacodynamic studies measure ROS levels and antioxidant enzyme activities in tissues.

Metabolism / Metabolites
This study employed gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) combined with S-band electron spin resonance (ESR) to investigate the metabolism of nitro oxygen radicals with different piperidine structures as spin markers in the human keratinocyte cell line HaCaT. Besides the known reduction of nitro oxygen radicals to ESR without signal hydroxylamine as the major product, our results indicate the formation of corresponding secondary amines. These reduction reactions were inhibited by the thiol blocker N-ethylmaleimide and the potent inhibitors of thioredoxin reductase (TR), 2-chloro-2,4-nitrobenzene and 2,6-dichloroindophenol. The competitive TR inhibitors azelaic acid and the cytochrome P-450 inhibitor metheprone had no such effect. The rates of reduction to hydroxylamine and secondary amines depended on the lipid solubility of the nitro oxygen radical. Therefore, it can be inferred that nitro oxides must enter the cell for bioreduction to occur. Intracellular nitrooxide reductants (such as ascorbic acid and glutathione) are the two most discussed examples, but neither can generate secondary amines. In summary, our results indicate that secondary amines are among the major metabolites of nitrooxides in keratinocytes, besides hydroxylamine, which is likely generated via flavin thioredoxin reductase. We also detected further metabolic transformations using benzoates of 4-oxo-2,2,6,6-tetramethylpiperidin-1-oxy and 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxy as substrates.
Tempol has a molecular formula of C9H18NO2 and a molecular weight of 172.25. It is a stable nitroxide radical that is soluble in water and organic solvents. The compound is stored at room temperature. It is administered orally or intraperitoneally in preclinical studies. Pharmacokinetic parameters including half-life, bioavailability, and tissue distribution are documented in research publications.

Interactions
Carrageenan can lead to increased reactive oxygen species (ROS) production, thereby promoting the pathophysiological process of inflammation. We investigated the effect of the membrane permeability free radical scavenger temozolomide (tempol) on carrageenan-induced pleurisy in rats. Rats were administered temozolomide at doses of 10, 30, or 100 mg/kg 15 minutes before carrageenan injection. The results showed that temozolomide dose-dependently reduced carrageenan-induced pleural effusion and polymorphonuclear cell migration. Temozolomide also reduced carrageenan-induced lung injury (histological examination) and the elevated levels of myeloperoxidase and malondialdehyde in lung tissue. However, temozolomide did not inhibit the activity of inducible nitric oxide synthase in lung tissue. Immunohistochemical analysis showed positive staining for nitrotyrosine in the lung tissue of carrageenan-treated rats. Lung tissue sections from carrageenan-treated rats also showed positive staining for poly(ADP-ribose) synthase (PARS). In tissue sections of carrageenan-treated rats treated with temozolomide (100 mg/kg), the staining intensity of nitrotyrosine and PARS was significantly reduced. Furthermore, temozolomide treatment significantly reduced (i) the production of peroxynitrite, (ii) DNA damage, (iii) impairment of mitochondrial respiratory function, and (iv) the decrease in NAD(+) cell levels observed in carrageenan-treated rat pleural macrophages. Temozolomide also alleviated cellular damage to cultured human endothelial cells caused by hydrogen peroxide (1 mM). This study is the first to demonstrate that tempol, a small molecule that can penetrate biological membranes and scavenge reactive oxygen species, can reduce the degree of inflammation and tissue damage associated with carrageenan-induced pleurisy in rats…
Acute pancreatitis occurred in rats 3 hours after intravenous infusion of secretin (10 μg/kg/hr). Autopsy findings of the pancreas under light microscopy revealed interstitial edema, vacuolar cell vacuolation, and leukocyte aggregation in pancreatic capillaries; electron microscopy revealed severely damaged mitochondrial and zymogen granule structures. Particularly observed were mitochondrial swelling and cristae breakage, as well as large vacuoles formed by the fusion of prozymogen granules and liposomes. Lipid peroxide levels in pancreatic tissue were significantly elevated. This study aimed to evaluate the role of the low molecular weight superoxide dismutase mimic 4-hydroxy-TEMPO in a rat model of acute pancreatitis, expecting it to significantly reduce free radical-mediated hydrogen peroxide production and tissue damage. Twenty-one male Wistar rats were divided into three groups: Group 1 (n=5) served as the control group, receiving intravenous infusion of normal saline for 3 hours; Group 2 (n=8) received intravenous infusion of secretin 10 μg/kg/h for 3 hours; and Group 3 (n=8) received simultaneous intravenous infusion of secretin and 4-hydroxy-TEMPO 22.6 mg/kg/h. Pancreatic tissue damage was quantified by measuring lipid hydrogen peroxide (LOOH) levels, organ weight, and light and electron microscopy observations. The concentration of 4-hydroxy-TEMPO in pancreatic tissue samples and pancreatic juice was determined by electron spin resonance (ESR) spectroscopy, and the cross-sectional area of 4-hydroxy-TEMPO was quantitatively analyzed. Injection of 4-hydroxy-TEMPO into rats prevented the formation of lipid peroxides and severe morphological damage. 4-hydroxy-TEMPO can cross the cell membrane barrier and be excreted into pancreatic juice. Infusion of 4-hydroxy-TEMPO appears to prevent free radical-induced pancreatic damage in experimental pancreatitis. Hemorrhagic shock (HS) is associated with the production of reactive oxygen species, which can lead to delayed multiple organ failure and death. Previous studies have shown that the antioxidant Tempol can improve physiological parameters of septic shock and HS, but not necessarily improve prognosis. We hypothesized that the combined use of free Tempol and Tempol bound to polynitroalbumin (PNA) (which has a prolonged half-life and reduced toxicity) could improve the prognosis of hemorrhagic shock (HS) in rats. In Study 1, HS was induced by drawing 3 mL/100 g of blood over 15 minutes. Mean arterial pressure was maintained at 40 mmHg by infusion of normal saline or blood aspiration for 20 to 90 minutes. Resuscitation (90–270 minutes) was performed by infusion of autologous blood. The observation period was 72 hours. At 45 minutes after the onset of hemorrhage (HS), albumin (ALB) (n = 10) or PNA + Tempol (n = 10) (1 mL/100 g/h) was slowly infused until 120 minutes. Study 2 was the same as Study 1 (n = 6 per group) but terminated at 150 minutes. Study 3 was the same as Study 1 but started at 20 minutes with either ALB or PNA + Tempol (n = 7 per group). The primary endpoint for Studies 1 and 3 was survival, while the primary endpoint for Study 2 was antioxidant reserves in serum and small intestine (the ability of serum or tissue homogenate to scavenge peroxy radicals generated by 2,2'-azobis[2-aminodipropane]dihydrochloride) and low molecular weight thiols in tissues (liver, small intestine, and kidney). In Study 1, the 72-hour survival rate was 1/10 (ALB group) and 2/10 (PNA+Tempol group). At 90 minutes, the pH value in the ALB group was lower than that in the PNA+Tempol group (p=0.02), and the pH value remained lower. Arterial blood lactate levels increased to 8.9 ± 3.2 mmol/L (mean ± standard deviation), compared to 6.5 ± 1.8 mmol/L in the control group (p = 0.04); base excess was -9.6 ± 4.3 mmol/L and -5.2 ± 3.2 mmol/L, respectively (p = 0.01) (ALB group vs. PNA + Tempol group). In Study 2, serum antioxidant reserves in the ALB group were lower than those in the PNA + Tempol group (p = 0.002). There were no differences between the two groups in small intestinal antioxidant reserves or in the levels of low molecular weight thiols in the liver, kidneys, and small intestine. In Study 3, the 72-hour survival rate was 0/7 in the ALB group and 5/7 in the PNA + Tempol group (p = 0.02). In Studies 1 and 3, the heart rate and systolic blood pressure in the ALB group were higher than those in the control group in the later stages of HS (p < 0.05). Early infusion of PNA + Tempol in HS improved survival. Late infusion of PNA + Tempol significantly improved acid-base balance and serum antioxidant status, but had no effect on survival…
The toxicity profile of tempol is characterized in preclinical safety studies. The compound is generally well-tolerated at therapeutic doses. Common adverse effects may include methemoglobinemia at high doses. The compound is for research use only and not for human use. Appropriate safety precautions should be taken during handling. The safety margin is determined from toxicology studies.

[1]. Resveratrol Reverses Functional Chagas Heart Disease in Mice. PLoS Pathog. 2016 Oct 27;12(10):e1005947.

[2]. Tempol, a membrane-permeable radical scavenger, reduces oxidant stress-mediated renal dysfunction and injury in the rat. Kidney Int. 2000 Aug;58(2):658-73.

Therapeutic Uses
Whole-brain radiotherapy commonly results in hair loss as a complication, exacerbating patient anxiety. Tempol, a nitrooxide radioprotective agent, has been shown in animal models to prevent radiation-induced hair loss. This Phase Ib study aimed to evaluate the safety and side effects of topical Tempol application in patients with brain metastases undergoing whole-brain radiotherapy. Between October 2000 and February 2003, 12 patients with brain metastases were enrolled. During 10 whole-brain radiotherapy sessions, Tempol (70 mg/mL solution) was applied topically to the scalp 15 minutes before each session and washed off immediately after treatment. Pharmacokinetic studies were also conducted to assess systemic absorption of Tempol. Toxicity was assessed before, during, and after Tempol administration. Additionally, the secondary endpoint of hair retention was scored. Eleven patients received topical Tempol treatment. Adverse events possibly, likely, or certainly associated with Tempol included: two cases of asymptomatic grade 2 hypoglycemia and one case of grade 1 hypoglycemia, one case of grade 1 frontal skin redness, one case of grade 1 scalp dryness, and one case of grade 1 scalp tingling. Tempol was undetectable in blood samples from over 50% of patients. The mean highest Tempol concentration at any time point ranged from 0.4 to 3.1 μmol/L for each patient. In the first four patients in the study, hair retention was observed at the scalp roots, the site of accumulation after Tempol solution application. Subsequently, in the last five evaluable patients, intact scalp hair retention was observed in three cases after the head was wrapped with gauze to secure the solution to the scalp. This study demonstrates that topical application of Tempol to the scalp prior to whole-brain radiotherapy is safe and well-tolerated. The study observed a protective effect of the product against radiation-induced hair loss. A phase II clinical trial using a gel formulation to increase scalp exposure to Tempol has been initiated.
Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) is a stable nitroxide radical with superoxide dismutase (SOD)-mimetic activity. It is a redox-active compound that scavenges reactive oxygen species and has been investigated for its antioxidant, anti-inflammatory, and radioprotective properties. Tempol reduces oxidative stress in various models of disease and injury. It is used for research purposes only.

C9H18NO2

172.2447

172.133

2226-96-2

Tempol-d17,15N;90429-66-6;Tempol-d17;100326-46-3

137994

Yellow to orange solid powder

1.187 g/cm3

269ºC

69-71 °C(lit.)

1.283

1

2

0

12

159

0

UZFMOKQJFYMBGY-UHFFFAOYSA-N

InChI=1S/C9H18NO2/c1-8(2)5-7(11)6-9(3,4)10(8)12/h7,11H,5-6H2,1-4H3

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~5.56 mg/mL (~32.28 mM)

Solubility in Formulation 1: 25 mg/mL (145.15 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)