Chemical name:	3-Carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyl-N-oxyl
Abbreviated name:	3CxP
Synonym:	3-Carboxy-PROXYL, 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyl-N-oxy, 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidin-1-yl--N-oxy free radical, PCA, 3-CTPY
Agent category:	Small molecule
Target:	Other
Target category:	Other-reactive oxygen species (ROS)
Method of detection:	Electron paramagnetic resonance imaging (EPRI), magnetic resonance imaging (MRI), proton electron double resonance imaging (PEDRI), Overhauser-enhanced MRI (OMRI)
Source of signal/contrast:	Nitroxide radicals
Activation:	No
Studies:	In vitro Rodents Non-primate non-rodent mammals	Click on the above structure for additional information in PubChem.

[PubMed]

Reactive oxygen species (ROS) are various free radicals generated in a biological milieu (1, 2). They are propagated through a cascade of reactions in the pathogenesis in many diseases, including cancer, stroke, atherosclerosis, ischemia-reperfusion injury, Alzheimer’s disease, diabetic vascular diseases, and inflammatory diseases (2). In particular, ROS interact with glutathione (GSH), NADPH, and ascorbates to maintain cellular redox status (3). Therefore, the distribution of ROS in tissue can be used as a surrogate marker to characterize the redox status/environment in disease-related physiological and pathological conditions (1). Because all free radicals contain unpaired electrons, the electron paramagnetic resonance (EPR) technique, also called electron spin resonance (ESR,) is specific for detecting and quantifying ROS (2). EPR spectra can provide a wealth of information for unequivocal identification of free radicals, such as fine, hyperfine, and superhyperfine structures, g-factor, and lineshape (2). EPR imaging (EPRI) technique allows for non-invasive mapping of free radicals in animals/organs (4).

EPR is fundamentally similar to nuclear magnetic resonance (NMR) (5). However, the differences in the physical and chemical properties of the resonance species (unpaired electrons versus nuclear spin) lead to three major differences in acquiring the spectra/images: gyromagnetic ratio, relaxation time, and concentration (5). The gyromagnetic ratio of an electron spin is 658 times larger than that of a proton nuclear spin, resulting in a 658-fold increase in its magnetic moment and resonant frequency. For instance, with a magnet of 0.34 T, the EPR frequency of X-band is 9.5 GHz, and the NMR frequency of proton nuclei is 14.4 MHz. As a result of strong non-resonant water absorption, a high radiofrequency such as 9.5 GHz is not suitable for examining tissue samples. Thus, much lower EPR frequencies in the range of 1.2 GHz (L-band) to 300 MHz are used instead, corresponding to a penetration depth of a few cm. The increase in the magnetic moment of electron spin provides ~700 times greater intrinsic sensitivity with EPR on a molar basis than with NMR. Because the excited electron spins relax on a nanosecond time scale, which is several orders of magnitude shorter than the nuclear spin (measured in ms), pulsed EPR (Fourier transformation EPR (FT EPR) or time-domain EPR) is only applicable to those free radicals with an extremely narrow line, whereas most ERP spectrometers use the continuous wave technique (CW EPR). The lack of high concentrations of naturally occurring paramagnetic species such as free radicals often requires the addition of paramagnetic species. This in turn allows for the quantification of exogenous paramagnetic species but also requires the acquisition of anatomic information with different imaging modalities such as magnetic resonance imaging (MRI). Proton electron double resonance imaging (PEDRI), also called Overhauser-enhanced magnetic resonance imaging (OMRI)) is a double resonance technique that encodes characteristic EPR spectral information on a high-resolution MRI (6). This method uses EPR irradiation to saturate paramagnetic species and leads to polarization of water protons through the dynamic nuclear polarization (DNP) effect. The polarized protons produce enhanced signal intensity in MRI. PEDRI offers good sensitivity, high spatial resolution, and signal enhancement of approximately two orders of magnitude (7).

Nitroxides are stable organic free radicals that have a single unpaired electron delocalized between the nitrogen and the oxygen (8). The steric hindrance around the nitroxide group makes these compounds very stable. They can be obtained in pure form, and they can be stored and handled in the laboratory with no more precautions than most organic substances (9). Nitroxides used as the contrast agent in EPRI can detect the redox status on the basis of their reduction to EPR-silent hydroxylamine (10), and nitroxides have been extensively used in cells, tissues, and living animals (11). Inside cells, nitroxides are reduced to hydroxylamine by cellular antioxidants such as ascorbate, thioredoxin, reductase, ubiquinol, NADPH and GSH. Nitroxides also can function as superoxide dismutase mimics and repair DNA damage caused by ultraviolet irradiation. In addition to the use as an EPRI contrast agent, nitroxides are T₁ relaxation agents in MRI for having an unpaired electron (12). Because their reduced form, hydroxylamine, is diamagnetic, the reduction process is accompanied by a decrease in T₁ relaxivity. This decrease reflects the alterations in the redox status and can be used to map the redox status. Although the T₁ relaxivity of nitroxides is much lower than that of gadolinium chelates (one unpaired electron versus seven unpaired electrons), their high cellular permeability leads to a significantly greater volume distribution in tissues and compensates for their lower relaxivity (12). Various nitroxides are designed to target different cellular compartments (8). For example, a neutral nitroxide can be distributed throughout the intracellular and extracellular environments, whereas a charged nitroxide is unable to cross the plasma membrane and can be used to measure oxygen levels in extracellular compartments. 3-Carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyl-N-oxyl (3CxP) is a charged nitroxide that is available commercially. This nitroxide is unable to permeate the cell and exhibits minimal lipophilicity. As a piperidine nitroxide with a line width of 1.4 Gauss and low toxicity, 3CxP is suitable for EPRI and Overhauser-enhanced magnetic resonance imaging in intact animals (11).

[PubMed]

3CxP was synthesized in several steps (13). First, acetone was condensed with ammonia in the presence of calcium chloride. The produced 4-oxo-2,2,6,6-tetramethylpiperidine (triacetoneamine) was converted to the corresponding nitroxyl radical (4-oxo-2,2,6,6-tetramethylpiperidinoxyl) by reaction with 30% hydrogen peroxide (14). The obtained nitroxyl radicals were then reacted with iodine in an alkaline medium to produce 2,2,5,5-tetramethylpyrrolinoxyl, which was hydrolyzed into 3CxP by hydrolysis (14).

[PubMed]

The T₁ relaxivity of 3CxP was determined to be ~0.2 mM^-1s^-1 (12). The partition coefficient of 3CxP in n-octanol/water was found to be 0.0047 (12).

[PubMed]

Sano et al. reported the biodistribution of 3CxP in mice (4–5 weeks old, 17–23 g) (15). After intravenous injection of 100 μl of 150 mM 3CxP solution, blood was collected 3 min later and tissues were rapidly harvested and homogenized, including brain, liver, kidney, heart, lung, spleen, stomach, and muscle. Blood or homogenates were transferred into capillary tubes and observed with an X-band EPR spectrometer (9.4 GHz). The distribution of 3CxP in tissue (μmol spin/gram of tissue) was found to be 0.0 ± 0.0 in brain, 1.18 ± 0.06 in blood, 0.08 ± 0.02 in liver, 0.51 ± 0.21 in kidney, 0.28 ± 0.13 in heart, 0.39 ± 0.07 in lung, 0.27 ± 0.05 in spleen, 0.12 ± 0.03 in stomach, and 0.12 ± 0.06 in muscle. The distribution of 3CxP plus its reduced form (hydroxylamine) in tissue (μmol spin/gram of tissue) was found to be 0.02 ± 0.0 in brain, 1.27 ± 0.08 in blood, 1.15 ± 0.28 in liver, 1.65 ± 0.27 in kidney, 0.72 ± 0.04 in heart, 0.42 ± 0.07 in lung, 0.47 ± 0.04 in spleen, 0.16 ± 0.02 in stomach, and 0.18 ± 0.06 in muscle. Sano et al. also examined the in vivo distribution of 3CxP in mouse brain (15). After intravenous injection, two-dimensional images were collected with an L-band ESR-computed tomography system; in these images the signal of 3CxP appeared only in the extracranial domain. Okajo et al. measured the pharmacokinetics 3CxP in rats (8 weeks old) (16). After intravenous injection of 3CxP at a dose of 0.3 mmol/kg, pharmacokinetics were obtained with a blood circulation monitoring method using an X-band EPR spectrometer (9.4 GHz). The EPR signal decay in blood exhibited a biphasic profile with α-phase rate constant k₁ of 0.444 ± 0.064 min^-1 and β-phase rate constant k₂ of 0.094 ± 0.0121 min^-1.

Hyodo et al. used MRI to study the in vivo pharmacokinetics of 3CxP in tumors (12). C3H mice (6–8 weeks old, 20–30 g) were implanted with squamous cell carcinoma (SCCVII) in the right hind leg. After intravenous injection of 3CxP at a dose of 1 mmol/kg (150 mM), T₁-weighted images were collected on a 4.7-T MRI imager. Maximum MRI intensity in the tumor area appeared at 9.3 min after injection, whereas the kidney exhibited continuous enhancements up to 17.8 min. There was no significant difference in the decay rate between the tumor (0.020 ± 0.014 min^-1) and the normal leg (0.029 ± 0.014 min^-1). The blood and organs were collected at 2.5, 7.5, 10, 15, and 20 min after injection, and the concentration of 3CxP in tissues was assessed with the use of an X-band EPR spectrometer. The levels of nitroxide plus nitroxylamine as a function of time did not demonstrate significant change during the imaging time in either tumor or muscle. Li et al. examined the distribution of 3CxP in mice (35 g) by PEDRI (11). After intravenously injecting 0.5 ml of 100 mM 3CxP solution, PEDRI was performed on a 0.38-T MRI imager (856 kHz NMR resonant frequency and 567 MHz EPR resonant frequency). 3CxP was found to distribute initially in the heart, lungs, major vessels, and kidneys. The image intensity peaked at ~2.5 min in the heart, lungs, and major vessels and at 5–10 min in the kidney, but returned to the preinjection levels at 30 min.

[PubMed]

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[PubMed]

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[PubMed]

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EB004900, EB00890, EB00306, EB00254