Radiation exposure is often undesirable, because it promotes carcinogenesis and toxicity in otherwise healthy tissue. Exposure can occur unexpectedly during a nuclear attack or power plant meltdown, but can also occur in a controlled medical environment during cancer radiotherapy. In each of these situations, countermeasures that protect radiosensitive organs from radiation damage may improve the outcome of the exposed individuals. In the current study, the nitroxide 23c was identified as an effective in vivo radioprotector, with an in vivo dose modification factor of 1.45 at the LD50/30 dose. To the authors’ knowledge, 23c is currently the most potent known nitroxide radioprotector.
A unique feature of nitroxide radioprotectors is that their pharmacokinetics can be measured non-invasively with magnetic resonance imaging (MRI). Using MRI, the pharmacokinetics of 22c and 23c were measured in the blood, liver, submandibular salivary gland, anterior leg muscle, oral muscle, brain, kidney, and tongue. It was found that both the peak concentration and rate of nitroxide metabolism and clearance varied substantially between tissues. However, for a given tissue, the peak concentration and reduction rate constant were usually the same for 22c and 23c. Additionally, due to the high BBB permeability of 23c, experiments were conducted that demonstrate the utility of 23c as a brain redox imaging contrast agent.
A useful question in radioprotection research is: “for a given set of radioprotectors, is there a correlation between the pharmacokinetic behavior of the radioprotector and the biological properties of the radioprotector?” For example, identification of correlations between peak tissue concentration on the one hand and toxicity or radioprotective potency on the other may lead to insights that will help to identify less toxic and more effective radioprotectors. The current study and a previous study [32
] together provide complete pharmacokinetic, radioprotection, and toxicity data for the nitroxides Tempol, 3-CP, 22c, and 23c. Based on these data, it is possible to identify or dispel correlations between the pharmacokinetic and biological properties of the nitroxides. It should be noted that although the discussion below is valid for the four nitroxides studied thus far, the correlation may not hold if additional nitroxides are considered. ranks the nitroxides Tempol, 3-CP, 22c, and 23c in the order in which they exhibit various biological or pharmacokinetic properties. For example, the row labeled “a” ranks the four nitroxides in order of their maximum tolerable dose during i.p. injection of the nitroxide (MTD). As can be seen from , the nitroxides fell in the order of 23c < 22c < Tempol < 3-CP in terms of their MTD. Notably, the order of MTD (, row a) exactly followed the order of normalized brain dose (, row c; the normalized dose for a given nitroxide is the peak brain concentration divided by the injected dose). That is, nitroxides with higher normalized brain dose were found to be more toxic than nitroxides with lower normalized brain doses. This observation, taken with the additional observation that overdosed mice die from seizure, implicates blood brain barrier permeability as a factor in nitroxide toxicity. Interestingly, the rate of nitroxide reduction by the brain (, row e) did not correlate with toxicity.
summary of toxicity, radioprotection, in vivo reduction rates, and in vivo peak tissue concentrations of four nitroxides.
Another important conclusion from is that the radioprotective potency of each nitroxide (row “b”) did not correlate with its whole-body peak tissue concentration (row “d”). The whole-body peak tissue concentration was calculated separately for each nitroxide by taking the average peak tissue concentration (, ref [32
]) of the submandibular salivary gland, tongue, anterior leg muscle, liver, brain, and kidney. The whole-body peak tissue concentration thus reflects the average accumulation of nitroxides in six different tissues. As can be seen from row “d”, the normalized whole-body peak nitroxide concentrations are roughly the same for all four nitroxides, and the whole-body concentration metric therefore cannot account for differences in radioprotective potency. Because 23c is the most potent radioprotector, the current study suggests that the variations in radioprotective potency were not determined by systematic differences in nitroxide accumulation in tissue. Thus, at first sight there might seem to be a discrepancy between the nearly identical pharmacological data of 22c and 23c and the significantly different in vivo
DMF of the two drugs.
This apparent discrepancy may be partly resolved by noting that the mice in the current study died of bone marrow toxicity. In general, the cause of death of mice exposed to whole body irradiation can be deduced from the time-course of mouse survival and the total whole-body dose. In terms of the survival time-course, death from cerebrovascular syndrome occurs usually within 24–48 hours, death from gastrointestinal syndrome occurs between 3–4 days, and death from hematopoetic syndrome (bone marrow death) peaks between 10–15 days after exposure [3
]. In the current study, of the 100 mice treated with 23c, 67 died of radiation exposure, and of those that died, 58 (85%) died between 7–15 days after exposure (data not shown). In terms of whole-body dose, 7 Gy results in 50% of mice dying from bone marrow toxicity before 30 days after exposure (i.e., the LD50/30
for mice is 7 Gy) [3
]. In the current study, the LD50/30
of the untreated control mice was 7.9 ± 0.15 Gy, which is suggestive of bone marrow toxicity. Thus, the predominant cause of death in the current study was apparently bone marrow toxicity, suggesting that in the surviving mice, 23c acts primarily by protecting the bone marrow.
Based on the conclusion that 23c protects mice from radiation-induced bone marrow toxicity, an attempt was made at measuring the pharmacokinetics of 22c and 23c in the bone marrow with MRI. The largest regions of marrow that could be identified were in the iliac crest and femur, but the size of these marrow compartments is at best comparable to the maximum resolution of the T1 weighted MRI scan, and it was therefore not possible to reproducibly measure the 23c and 22c levels in the bone marrow using MRI. Nonetheless, the survival timeline implicates the bone marrow as the site of 23c radioprotection over the radiation dose range studied (7–12 Gy).
The substantial difference in DMF between 16c, 22c, and 23c implies a structure-activity relationship for in vivo
nitroxide radioprotection. An in vivo
structure-activity study is well beyond the scope of this study, but it is worth speculating about what factors may connect the molecular structure of these nitroxides to their in vivo
DMF. In theory, the in vivo
DMF of nitroxides may be influenced by their subcellular compartmentalization and/or metabolism. In terms of compartmentalization, DNA damage is a major cause of radiation toxicity [3
], and it follows that the degree to which a nitroxide non-covalently associates with DNA may affect its DMF. Association of nitroxides with DNA may occur due to residual positive charge on the nitroxide, which would promote an electrostatic attraction between the nitroxide and negatively charged DNA. In the case of 16c, 22c, and 23c, the nitrogen atoms on the pyrroline ring substituent exhibit a residual positive charge, which may promote their association with DNA. However, 16c, 22c, and 23c each have a residual charge on the nitrogen, and so the existence of a positive charge alone cannot account for the difference in the in vivo
DMFs of these three nitroxides. The difference between the DMF of 22c and 23c might be explained by the molecular structure of the positively charged ring substituent. For 22c, the substituent is the relatively planar N-methyl pyrrolidine group, while the substituent for 23c is the non-planar N-methyl piperidine. Because the non-planar substituent of 23c is capable transforming between “boat” and “chair”-like conformations, both with a protruding positively charged nitrogen atom, 23c may form a stronger attractive electrostatic interaction with DNA than 22c. This may result in greater proximity of 23c than 22c with DNA molecules, which may contribute to the greater in vivo
DMF of 23c. This theory does not, however, explain why 16c and 22c are potent radioprotectors in vitro
, but not in vivo
The difference between the in vivo and in vitro DMFs of 16c, 22c, and 23c may be due to enzymatic metabolism of these nitroxides in vivo. That is, 23c may be metabolized by yet to be identified enzymatic reactions into a radioprotective molecule, or alternatively, 16c and 22c may be metabolized into non-radioprotective species. Because many enzymes catalyze chemical reactions in a kinetically rapid and substrate-specific manner, the involvement of enzymes in the metabolism of these three nitroxides may have caused the observed differences in their in vivo DMFs.
Because 23c readily passes the blood brain barrier (BBB), 23c was evaluated as brain redox imaging agent. To be a viable redox imaging agent, a nitroxide must accumulate in measurable quantities in the target organ, and its rate of washout must be substantially less than the rate of reduction. If these conditions are met, then it is reasonable to assume that signal decay constants are informative of tissue redox status. With regards to accumulation of 23c in the brain, it was found that the peak concentration of 23c in the brain was 3.6 ± 0.4 mM, which provided more than sufficient contrast on T1 weighted images. With regards to washout, it was found that over a 3 minute period washout alone (measured invasively) accounted for only 40% of signal loss, while washout and reduction together (measured with MRI) resulted in 90% signal loss over the same period (). These data suggest that the signal decay constant of 23c in the brain is related to the redox status of the tissue.
Using 23c as a brain redox imaging agent, spatial variations in brain redox status were identified. In particular, the ventral regions of the brain exhibited a decay constant kr
that was 50% greater than surrounding brain regions (). The regions with a more reducing environment were the spinal cord, thalamus, hypothalamus, and lower midbrain. Spatial variations in brain redox status are not surprising given that metabolic rates and antioxidant capacity varies spatially within the rodent brain. For example, spatial variations in glutathione levels within the rodent brain have been noted [34
], and glutathione is known to influence the rate of nitroxide reduction in vivo
]. Additionally, spatial variations in the rate of glucose metabolism have been observed in the rodent brain [37
]. Because glucose can feed into the pentose phosphate pathway (PPP), and because the rate of nitroxide reduction correlates with PPP activity in vitro
], the rate of glucose metabolism may correlate with the rate constant of 23c reduction in mice.
The ability of 23c to provide redox data in the mouse brain may prove to be useful in future studies, because many neurocognitive disorders and stressors are associated with oxidative stress. Examples include sleep deprivation [35
], MDMA (Ecstasy) neurotoxicity [42
], and phencyclidine (PCP)-induced schizophrenia [46
]. Furthermore, in the MDMA exposure [45
] and schizophrenia [46
] models, the levels of oxidative stress varied between different brain structures. Because nitroxide imaging allows spatial mapping of tissue redox status with high resolution (~0.2×0.2×1 mm) (Figure 9 and refs [32
]), the novel nitroxide 23c may allow brain-region specific assessment of oxidative stress in preclinical models of neurocognitive damage.
An unexpected observation made in the current study was that high levels of 23c and 22c briefly accumulated in the myocardium after injection (23c is shown in ). This was unexpected, because in a previous study, myocardial accumulation was not observed for the nitroxides Tempol and 3-CP [32
]. Further experiments are necessary to better understand the mode of 23c delivery (e.g. via the ventricle or via the coronary arteries) and the cause of 23c signal decay (e.g. clearance and/or reduction). Depending on the mode of delivery and cause of decay, 23c may allow localization of a thrombus in the coronary arteries and/or the resulting myocardial infarct.
In summary, the current manuscript reports several novel findings. First, it is reported for the first time that 23c is a potent in vivo nitroxide radioprotector. Second, the pharmacokinetics of 22c and 23c were characterized in various healthy tissues using magnetic resonance imaging. It was found that 22c and 23c exhibit almost identical pharmacokinetics in the tissues studied, with the important exception that 23c accumulated in the brain at a greater concentration than 22c. Third, it was found that the exponential decay rate of 23c in the mouse brain reflects the redox status of the underlying tissue, and that inherent redox differences exist in the mouse brain. Finally, the observation was made that 23c accumulates in the myocardium at high concentrations relative to other nitroxides, and that this effect may be useful for functional imaging of the mouse heart. These findings highlight the versatile biomedical applications of the nitroxide 23c.