NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.

Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2013.

Cover of Molecular Imaging and Contrast Agent Database (MICAD)

Molecular Imaging and Contrast Agent Database (MICAD) [Internet].

Show details

3-Carbamoyl-2,2,5,5-tetramethyl-1-pyrrolidinyl-N-oxyl

3CP
, PhD
National Center for Biotechnology Information, NLM, NIH, Bethesda, MD, vog.hin.mln.ibcn@dacim

Created: ; Last Update: June 9, 2008.

Chemical name:3-Carbamoyl-2,2,5,5-tetramethyl-1-pyrrolidinyl-N-oxylimage 24892761 in the ncbi pubchem database
Abbreviated name:3CP
Synonym:3-Carbamoyl-PROXYL, 3-carbamoyl-2,2,5,5-tetramethyl-1-pyrrolidinyl-N-oxy, 3-carbamoyl-2,2,5,5-tetramethyl-1-pyrrolidin-1-yl-N-oxy free radical
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:
  • Checkbox In vitro
  • Checkbox Rodents
  • Checkbox Non-primate non-rodent mammals
Click on the above structure for additional information in PubChem.

Background

[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 the presence 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 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. 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-Carbamoyl-2,2,5,5-tetramethyl-1-pyrrolidinyl-N-oxyl (3CP) is a neutral nitroxide that is available commercially. As a piperidine nitroxide with a line width of 1.4 Gauss and low toxicity, 3CP is suitable for EPRI and Overhauser-enhanced magnetic resonance imaging in intact animals (11).

Synthesis

[PubMed]

3CP 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 treated with iodine in an alkaline medium to produce 3CP (14).

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

The T1 relaxivity of 3CP was determined to be 0.95 ± 0.15 mM-1s-1 in the low-field limit (<5 MHz) and decreased to 0.30 ± 0.05 mM-1s-1 at 50 MHz (15). The partition coefficient of 3CP in n-octanol/water was found to be 0.68 (16). 3CP in aqueous solution exhibited sharp triplet lines in L-band EPR spectrum (6) with hyperfine coupling constant of 14-15 G (14).

Animal Studies

Rodents

[PubMed]

Nishino et al. used EPRI to study the in vivo pharmacokinetics of 3CP in mice (n=4) and rats (n=4) at different ages (17). Mice (4, 6, 10, 17, 37, and 52 weeks old) and rats (3, 4, 5, and 10 weeks old) were injected intravenously with 3CP at a dose of 1 mmol/kg. An L-band EPR spectrometer (1.1 GHz) was used to measure the signal of radicals in the upper abdomen of the animals. The EPR signal decay of 3CP exhibited a biphasal profile with α-phase rate constant k1 of 0.126 ± 0.0042 min-1 and β-phase rate constant k2 of 0.066 ± 0.0012 min-1. In fresh mouse blood, 3CP remained stable with a half-life (t1/2) of 1,286 ± 176 min. Li et al. used PEDRI to examine the distribution of 3CP in mice (35 g) (11). After intravenously injecting 0.5 ml of 100 mM 3CP solution, PEDRI was performed on a 0.38-T MRI imager (856 kHz NMR resonant frequency and 567 MHz EPR resonant frequency). 3CP was found to distribute initially in the heart, lungs, major vessels, and kidneys. Image intensity peaked at ~5 min in these areas and decayed to preinjection levels at 16 min. Okajo et al. measured the pharmacokinetics of 3CP in rats (8 weeks old) (18). After intravenous injection of 3CP 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 showed a biphasic profile with k1 of 0.601 ± 0.043 min-1 and k2 of 0.0662 ± 0.0045 min-1.

Hyodo et al. used MRI to study the in vivo pharmacokinetics of 3CP in tumors (12). C3H mice (6–8 weeks old, 20–30g,) were implanted with squamous cell carcinoma (SCCVII) in the right hind leg. After intravenous injection of 3CP at a dose of 1.5 mmol/kg, T1-weighted images were collected on a 4.7-T MRI imager. The contrast in the tumor area increased until 2.8 min after injection and decreased after 7.8 min. The decay rate in the tumor (0.107 ± 0.020 min-1) was approximately two times higher than that in the normal leg (0.056 ± 0.013 min-1). The blood and organs were collected at 2.5, 7.5, 10, 15, and 20 min after injection, and the concentration of 3CP 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 in tumor or muscle during the imaging time. Matsumoto et al. compared the pharmacokinetics of 3CP in the same tumor model as measured with EPRI with that measured with MRI (6). EPR images were collected on a 300-MHz EPR imager, and T1-weighted MRI images were collected on a 4.7-T MRI imager. EPRI and MRI showed similar reduction rates of 3CP, which were 0.0783 ± 0.023 min-1 for normal leg and 0.0870 ± 0.0233 min-1 for tumor leg on EPRI, and 0.0669 ± 0.0108 min-1 for normal leg and 0.0973 ± 0.0090 min-1 for tumor leg on MRI. Mikuni et al. used EPRI to examine the pharmacokinetics of 3CP in gastric carcinoma in rats (4). Rats (n = 8) with gastric cancer tumors (2–8 mm in diameter) or healthy rats (n = 6) as control were injected intravenously with 1.5 ml of 300 mM 3CP solution. EPR images were collected on a 750-MHz EPR imager after injection. The level of 3CP in the tumors was too low to be detected compared with that in the normal stomach tissue (t1/2 = ~8.7 ± 0.6 min).

Velayutham et al. used EPRI to examine the distribution of 3CP in rat hearts (19). The hearts of rats (140–160 g) after occlusion of the left anterior descending coronary artery were perfused with cardioplegic buffer containing 1.5 mM of 3CP. EPR images of the hearts were collected with a L-band EPR spectrometer (1.2 GHz). A loss in signal intensity was found in the ischemic region, which was further confirmed with histological measurements. Hirayama et al. used EPRI with 3CP to examine various phases of murine renal ischemia-reperfusion injury in mice deficient in NF-E2–related factors (3). After intravenous injection of 30 μmol 3CP, EPR images in the upper abdominal area were collected at 4, 6.5, 9, and 11.5 min. Compared with the pre-ischemia phase and the recovered phase, the injured phase demonstrated a remarkably prolonged decay in signal intensity.

Other Non-Primate Mammals

[PubMed]

The physiological effects of 3CP were tested in healthy pigs (6–12 month old, 30–80 kg) (20). 3CP was injected intravenously at doses of 30 mg/kg, 150 mg/kg, and 300 mg/kg with 4 days between doses. No changes in blood pressure or heart rate were noticed. No observable clinical toxicity was found.

Non-Human Primates

[PubMed]

No publication is currently available.

Human Studies

[PubMed]

No publication is currently available.

NIH Support

EB004900, EB00890, EB00306, EB00254, CA78886, RR12190, GM58582, NIH intramural research program

References

1.
Soule B.P. , Hyodo F. , Matsumoto K. , Simone N.L. , Cook J.A. , Krishna M.C. , Mitchell J.B. Therapeutic and clinical applications of nitroxide compounds. Antioxid Redox Signal. 2007;9(10):1731–43. [PubMed: 17665971]
2.
Kuppusamy P. , Zweier J.L. Cardiac applications of EPR imaging. NMR Biomed. 2004;17(5):226–39. [PubMed: 15366025]
3.
Hirayama A. , Nagase S. Electron paramagnetic resonance imaging of oxidative stress in renal disease. Nephron Clin Pract. 2006;103(2):c71–6. [PubMed: 16543759]
4.
Mikuni T. , He G. , Petryakov S. , Fallouh M.M. , Deng Y. , Ishihara R. , Kuppusamy P. , Tatsuta M. , Zweier J.L. In vivo detection of gastric cancer in rats by electron paramagnetic resonance imaging. Cancer Res. 2004;64(18):6495–502. [PubMed: 15374960]
5.
Gallez B. , Swartz H.M. In vivo EPR: when, how and why? NMR Biomed. 2004;17(5):223–5. [PubMed: 15366024]
6.
Matsumoto K. , Narazaki M. , Ikehira H. , Anzai K. , Ikota N. Comparisons of EPR imaging and T1-weighted MRI for efficient imaging of nitroxyl contrast agents. J Magn Reson. 2007;187(1):155–62. [PubMed: 17433743]
7.
Hyodo F. , Murugesan R. , Matsumoto K. , Hyodo E. , Subramanian S. , Mitchell J.B. , Krishna M.C. Monitoring redox-sensitive paramagnetic contrast agent by EPRI, OMRI and MRI. J Magn Reson. 2008;190(1):105–12. [PMC free article: PMC2258209] [PubMed: 18006345]
8.
Gallez B. , Baudelet C. , Jordan B.F. Assessment of tumor oxygenation by electron paramagnetic resonance: principles and applications. NMR Biomed. 2004;17(5):240–62. [PubMed: 15366026]
9.
Holtzman, J.L., ed. Spin labeling in pharmacology1984, Academic Press, INC: New York. 5.
10.
Kroll C. , Borchert H.H. Metabolism of the stable nitroxyl radical 4-oxo-2,2,6, 6-tetramethylpiperidine-N-oxyl (TEMPONE) Eur J Pharm Sci. 1999;8(1):5–9. [PubMed: 10072473]
11.
Li H. , He G. , Deng Y. , Kuppusamy P. , Zweier J.L. In vivo proton electron double resonance imaging of the distribution and clearance of nitroxide radicals in mice. Magn Reson Med. 2006;55(3):669–75. [PubMed: 16463344]
12.
Hyodo F. , Matsumoto K. , Matsumoto A. , Mitchell J.B. , Krishna M.C. Probing the intracellular redox status of tumors with magnetic resonance imaging and redox-sensitive contrast agents. Cancer Res. 2006;66(20):9921–8. [PubMed: 17047054]
13.
Sosnovsky G. , Konieczny M. Preparation of triacetoneamine (4-oxo-2,2,6,6-tetramethylpiperidine), an improved method. Synthesis. 1976:2.
14.
Zhdanov, R.I., Nitroxyl radicals and non-radical reactions of free radicals. Bioactive spin labels, ed. R.I. Zhdanov. 1992, New York: Springer-verlag. 26.
15.
Bennett H.F. , Brown R.D. 3rd, Koenig S.H. , Swartz H.M. Effects of nitroxides on the magnetic field and temperature dependence of 1/T1 of solvent water protons. Magn Reson Med. 1987;4(2):93–111. [PubMed: 3031423]
16.
Hyodo F. , Yasukawa K. , Yamada K. , Utsumi H. Spatially resolved time-course studies of free radical reactions with an EPRI/MRI fusion technique. Magn Reson Med. 2006;56(4):938–43. [PubMed: 16964613]
17.
Nishino N. , Yasui H. , Sakurai H. In vivo L-band ESR and quantitative pharmacokinetic analysis of stable spin probes in rats and mice. Free Radic Res. 1999;31(1):35–51. [PubMed: 10489118]
18.
Okajo A. , Matsumoto K. , Mitchell J.B. , Krishna M.C. , Endo K. Competition of nitroxyl contrast agents as an in vivo tissue redox probe: comparison of pharmacokinetics by the bile flow monitoring (BFM) and blood circulating monitoring (BCM) methods using X-band EPR and simulation of decay profiles. Magn Reson Med. 2006;56(2):422–31. [PubMed: 16810697]
19.
Velayutham M. , Li H. , Kuppusamy P. , Zweier J.L. Mapping ischemic risk region and necrosis in the isolated heart using EPR imaging. Magn Reson Med. 2003;49(6):1181–7. [PubMed: 12768597]
20.
Hahn S.M. , Sullivan F.J. , DeLuca A.M. , Bacher J.D. , Liebmann J. , Krishna M.C. , Coffin D. , Mitchell J.B. Hemodynamic effect of the nitroxide superoxide dismutase mimics. Free Radic Biol Med. 1999;27(5-6):529–35. [PubMed: 10490272]

Views

Search MICAD

Limit my Search:


Related information

Similar articles in PubMed

See reviews...See all...

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...