C4 (cobalt dichloride-N-acetylcysteine [1% CoCl2:2% NAC]) is a novel magnetic resonance imaging contrast marker that facilitates visualization of implanted radioactive seeds in cancer brachytherapy. We evaluated the toxicity of C4. Rats were assigned to control (0% CoCl2:NAC), low-dose (0.1% CoCl2:2% NAC), reference-dose (C4), and high-dose (10% CoCl2:2% NAC) groups. Agent was injected into the left quadriceps femoris muscle of the rats. Endpoints were organ and body weights, hematology, and serum chemistry and histopathologic changes of tissues at 48 hours and 28 and 63 days after dosing. Student’s
The current standard of care after brachytherapy for localized prostate cancer includes an image-guided quality assurance check of the radioactive seed placement and anticipated dose distribution. Historically the imaging technique used for this purpose is computed tomography (CT). Although CT can effectively visualize the seeds, it is less successful for visualizing soft tissues such as the prostate. Magnetic resonance imaging (MRI), on the other hand, is ideal for visualizing soft tissues [
Solutions at each dose level were prepared within 1 week of administration under a standard operating procedure in which CoCl2:NAC is prepared from cobalt chloride hexahydrate (CoCl2 • 6H2O; Sigma), N-acetylcysteine (C5H9NO3S; Sigma), and ultrapure, filtered, double-deionized water (Milli-Q, Millipore). Solutions were stored at 5 ± 3°C and protected from light until administration. At the prescribed injection times, solutions were warmed to room temperature and transferred to the animal facility while still protected from light. Solutions were analyzed for total cobalt content both before and after dosing by inductively coupled plasma mass spectrometry at A&B Labs (Houston, TX).
Sixty male Sprague-Dawley rats, aged 12-13 weeks and weighing 300–450 g, were supplied by Charles River Laboratories. This species has been accepted to support studies of compounds intended to be used in humans. Procedures for animal housing and care were carried out in accordance with the Animal Welfare Act as amended in the recommendations and guidelines of the Public Health Service, the US Department of Agriculture, the Association for Assessment and Accreditation of Laboratory Animal Care International, and Institutional Animal Care and Use Committee of UT MD Anderson Cancer Center. Environmental conditions were within specified limits in at least 90% of scheduled observations. Rats were housed up to 3 per cage with bedding or cage board (rather than cedar or pine chips), and cages were sanitized regularly. No known contaminants that could affect the results of the study were present in the bedding. The rats were fed a commercial, dry rodent chow ad libitum, and water was also given ad libitum. All rats were quarantined for at least 3 days before dosing, and no prophylactic or therapeutic treatments were given during the quarantine period. Rats were identified individually, by cage, and by treatment group.
Rats were assigned to one of four dosage groups (15 rats/dose group): control (0% CoCl2:NAC), low dose (0.1% CoCl2:2% NAC), mid- or reference dose (1% CoCl2:2% NAC; this is the solution used in the MRI-marker medical device), or high dose (10% CoCl2:2% NAC). These concentrations were based on the concentrations contained within the reference medical device (1% CoCl2 • 6H2O), bracketed by one higher level (10% CoCl2 • 6H2O) and one lower level (0.1% CoCl2 • 6H2O). All solutions contained 2% NAC. The solutions were given in 9-
Rats were observed for clinical signs daily and weighed twice weekly. At 2 days (48 hours), 28 days, or 63 days after the injection, 5 rats from each dose group (3 experimental and 1 control) were euthanized with carbon dioxide, weighed, exsanguinated, and subjected to necropsy. Blood samples were collected immediately after euthanasia via cardiac puncture for hematology and serum chemistry analyses from all 4 groups at each of the 3 time points. All rats were weighed just before terminal blood collection. At necropsy, the liver, kidney, and spleen were weighed, all organs were examined grossly, and tissue samples described in Supplemental Table
Blood samples collected in microtubes with serum separator were allowed to clot for 30 minutes after collection and were then centrifuged for 2 minutes at 17,000 rpm for isolating the blood serum that was processed for serum chemistry analysis. For hematologic analyses, blood samples were collected in microtubes containing ethylenediaminetetraacetic acid (EDTA); for the complete blood cell count analysis, blood smears were stained with Diff-Quik. Complete blood cell count analysis was performed with a Cobas Integra 400 Plus instrument (Roche) and serum chemistry measurements with an Advia 120 instrument (Siemens AG).
After fixation, all tissues (Tables
Hematologic findings.
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| 61.7 (57.8-64.0) | 30.8 (29.1-32.6) | 2.3 (1.04-2.99) | 18.4 (10.4-24.9) | 0.27 (0.10-0.43) | 2.1 (1.0-2.7) | 9.63 (6.76-11.84) | 76.6 (68.7-87.2) |
| 61.1 (57.1-63.3) | 30.9 (29.5-32.1) | 3.1 (1.67-4.63) | 21.6 (15.3-27.9) | 0.33 (0.25-0.56) | 2.3 (1.8-2.8) | 10.5 (8.43-15.38) | 72.5 (66.7-80.9) |
| 61.3 (57.8-62.9) | 30.1 (29.4-30.6) | 3.1 (0.9-5.27) | 20.1 (11.2-26.7) | 0.33 (0.09-0.54) | 2.1 (1.2-2.8) | 10.1 (6.84-15.13) | 74.7 (67.0-85.4) |
| 58.5 | 30.9 (30.2-31.7) | 3.5 (2.15-4.64) | 26.6 (19.0-34.4) | 0.4 (0.27-0.87) | 2.9 (2.0-5.4) | 8.7 (6.75-10.15) | 66.7 (58-75.3) |
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| 58.7 (58-59.6) | 30.5 (30.3-30.7) | 2.21 (1.03-2.87) | 22.1 (11.6-28.4) | 0.22 (0.16-0.3) | 2.2 (1.5-2.9) | 6.99 (6.31-7.39) | 71.7 (62.4-82.9) |
| 59.3 (56.5-62.0) | 30.5 (30.1-30.9) | 2.5 (1.31-3.95) | 21.6 (15-34.4) | 0.27 (0.18-0.35) | 2.5 (1.5-4.0) | 8.3 (6.23-13.28) | 71.6 (55.6-80) |
| 58.8 (57.3-59.8) | 31.1 (29.8-31.7) | 2.3 (1.08-3.57) | 24.2 (13.1-37.1) | 0.31 (0.16-0.56) | 31. (1.9-5.9) | 6.8 (5.26-9.78) | 69.4 (54.8-81.5) |
| 58.8 (56.5-60.1) | 30.6 (29.8-31.4) | 2.7 (2.07-3.20) | 23.1 (20.1-26.8) | 0.31 (0.20-0.53) | 2.7 (1.9-4.8) | 8.2 (7.05-10.87) | 70.4 (63.5-73.8) |
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| 58.8 (57.7-61.2) | 30.9 (30.6-31.4) | 1.65 (0.95-2.29) | 15 (8.9-20.3) | 0.22 (0.16-0.37) | 2 (1.3-3.3) | 8.83 (7.43-9.96) | 80.5 (73.2-87.0) |
| 58.9 (57.0-60.5) | 31.9 | 2.0 (1.16-2.52) | 16.2 (12.0-19.4) | 0.26 (0.19-0.35) | 2.2 (1.5-3.1) | 9.3 (8.03-10.75) | 78.5 (74.7-83.6 |
| 58.7 (57.2-60.7) | 32.2 | 1.8 (1.06-2.68) | 15.7 (10.1-24.2) | 0.23 (0.11-0.34) | 2.0 (1.3-3.2) | 8.8 (7.01-11.99) | 78.6 (69.6-85.3) |
| 57.8 (55.8-61.1) | 31.4 (30.1-32.3) | 1.6 (1.0-2.38) | 12.6 (6.9-16.8) | 0.25 (0.09-0.38) | 1.9 (0.9-2.7) | 10.4 (9.13-12.91) | 82.8 (78.0-89.9) |
Data are shown as mean (range).
Control, 0% CoCl2:NAC; Low dose, 0.1% CoCl2:2% NAC; Ref[erence] dose 1% CoCl2:2% NAC; High dose, 10% CoCl2:2% NAC.
MCV, mean corpuscular volume (in femtoliters); MCHC, mean corpuscular hemoglobin concentration.
Clinical chemistry findings.
| | | | | | | | | |
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| 0.1 (0.1) | 0.3 (0.28-0.32) | 15.80 (14.2-16.7) | 219 (122-445) | 175 (54-441) | 168 (98-228) | 6.18 (5.58-6.59) | 4.21 (4.01-4.60) | 1.97 (1.57-2.30) |
| 0.1 (0.1) | 0.3 (0.28-0.35) | 16.40 (14.8-18.8) | 283 (121-869) | 266 (78-921) | 208 (132-306) | 6.23 (5.83-6.74) | 4.16 (4.11-4.22) | 2.07 (1.72-2.61) |
| 0.1 (0.1) | 0.27 (0.24-0.30) | 16.9 (15.9-19.6) | 199 (68-491) | 173 (44-428) | 201 (175-239) | 6.1 (5.88-6.28) | 4.2 (4.09-4.35) | 1.9 (1.53-2.18) |
| 0.1 (0.1) | 0.29 (0.25-0.34) | 16.1 (14.0-18.8) | 132 (70-318) | 99 (44-244) | 167 (144-230) | 6.21 (5.82-6.44) | 4.04 (3.87-4.16) | 2.17 (1.95-2.45) |
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| <0.1 (<0.01) | 0.32 (0.28-0.35) | 17.9 (16.2-19.5) | – (154->3836)† | – (48->4070)† | 186 (141-252) | 6.3 (5.98-6.95) | 4.26 (4.17-4.47) | 2.04 (1.78-2.48) |
| <0.01 (<0.01) | 0.31 (0.27-0.34) | 20.1 (17.3-24.6) | – (123->964)† | – (65->921)† | 202 (173-258) | 6.36 (5.91-6.74) | 4.18 (3.95-4.36) | 2.17 (1.76-2.66) |
| <0.01 (<0.01) | 0.32 (0.29-0.37) | 12.2 (15.8-20.2) | – (56->2530)† | – (45->3030)† | 140 | 6.68 (6.34-6.83 | 4.19 (4.04-4.31) | 2.49 |
| <0.01 (<0.01) | 0.31 (0.31->0.69)† | 19.3 (17.4-21.4) | – (59->7000)† | – (45->7000)† | 210 (131-287) | 6.87 (6.39-7.87) | 4.32 (4.11-4.60) | 2.51 (2.13-3.27) |
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| 0.1 (0.1) | 0.34 (0.32-0.36) | 19.9 (18.8-21.4) | 135 (68-325) | 141 (41-473) | 141 (111-195) | 6.89 (6.56-7.21) | 4.33 (4.21-4.44) | 2.56 (2.21-2.88) |
| 0.1 (0.1) | 0.33 (0.31-0.35) | 20.7 (17.6-23.6) | 142 (84-266) | 113 (52-239) | 165 (127-206) | 7.01 (6.69-7.70) | 4.40 (4.14-4.90) | 2.61 (2.48-2.80) |
| 0.1 (0.1) | 0.32 (0.30-0.35) | 19.6 (17.9-21.7) | 247 (180-336) | 193 (153-230) | 149 (89-235) | 6.57 (6.26-7.10) | 4.22 (3.98-4.58) | 2.36 (2.00-2.52) |
| 0.1 (0.1) | 0.31 (0.26-0.33) | 18.4 (16.9-19.5) | 136 (67-232) | 114 (39-233) | 115 (100-135) | 6.73 (6.27-7.33) | 4.16 (3.89-4.57) | 2.57 (2.38-3.07) |
Data are shown as mean (range).
Control, 0% CoCl2:NAC; Low dose, 0.1% CoCl2:2% NAC; Ref[erence] dose, 1% CoCl2:2% NAC; High dose, 10% CoCL2: 2% NAC.
Each experiment was repeated at least three times. Unless otherwise noted, data are presented as means and ranges, and Student’s
All animals survived until their scheduled termination dates. No adverse clinical signs or abnormalities in body weight or in absolute or relative organ weights were noted, and no significant hematologic abnormalities were found (Table
Similarly, no significant abnormalities of serum chemistry tests were noted (Table
No gross pathologic lesions were found during necropsy, and no histopathologic evidence of acute or chronic systemic toxicity or inflammation was observed in any of the tissues examined (including brain, spinal cord, lung, heart, liver, kidney, and spleen) from the control and high-dose groups at 48 hours or 28 days after i.m. injection of CoCl2:NAC.
Acute dose-related injury of transient focal myotoxicity at the injection site was noted in all three treatment groups at 48 hours after injection (Figures
Histopathological changes of skeletal muscle at the site of C4 (CoCl2:NAC) injection. A volume of nine microliters of CoCl2:NAC at various concentrations (0% CoCl2:NAC [control], 1% CoCl2:2% NAC [mid- or reference dose], and 10% CoCl2:2% NAC [high dose]) was injected intramuscular into the middle region of the left hind limb quadriceps femoris muscle of male Sprague-Dawley rats. Images of H&E stained tissue sections at 20x and 40x magnification. (a)–(c) Images show mild inflammation (arrow) in control animals (a) and acute degeneration and necrosis of myofibers with granulomatous inflammation at the site of intramuscular injection (arrows) in the mid- and high-dose groups ((b) and (c), respectively), at 48 hours after treatment. (d)–(f) Images show minimal edema with occasional degenerated myofibers (arrow) in control (d) and middose group (e) animals, which is associated with focal mild lymphohistiocytic inflammation (arrow) and myofiber regeneration in the high-dose group (f), at 28 days after treatment. (g)–(i) Images reveal presence of rare small foci of histiocytic inflammation (arrow) similar in control and treated groups of animals ((g), (h), and (i)), and areas of normally regenerated myofibers surrounded by minimal interstitial fibrosis in the mid- and high-dose groups ((h) and (i), respectively), at 63 days after treatment.
In the chronic stages of treatment, at 28 and 63 days after injection, no degenerative or necrotic lesions were observed in the left quadriceps femoris in any animals from the control or treated groups (Figures
The results of this study of Sprague-Dawley rats indicated that a single intramuscular injection, in the middle of the quadriceps femoris muscle, of up to 10% CoCl2:2% NAC produced no significant treatment-related adverse clinical signs, no changes in body or organ weights, and no significant changes in hematologic or serum chemistry values. Similarly, postmortem pathological investigations revealed no gross or histopathologic evidence of acute or chronic systemic toxicity or inflammation. At 48 hours after i.m. injection, minor acute hematologic changes were noted in neutrophil, monocyte, and lymphocyte numbers, and mild focal myotoxicity manifested with acute degeneration and necrosis of myofibers associated with acute granulomatous inflammation at the injection site. Since these lesions healed and returned to normal skeletal muscle at 28 and 63 days after i.m. injection, these changes are considered to be a normal physiologic healing response to transient local acute muscle injury.
MRI provides superb anatomic resolution [
With regard to acute local response, we did find evidence of focal acute degeneration and necrosis of myofibers associated with acute granulomatous inflammation at the injection site 48 hours after the i.m. injection. One review [
Our study did have limitations. Not all of the biochemical markers that have been suggested as possible toxic indicators were included in this study, as sometimes it is impossible to plan for all measurements of toxicity when using small species.
In conclusion, we showed that a single intramuscular dose of the novel MRI-marker C4, in concentrations ranging from 0 to 10 times the dose intended for clinical use, produced no acute or chronic clinical systemic toxicity or inflammation in Sprague-Dawley rats. These results suggest that C4 may not cause acute or chronic clinical systemic toxicity or inflammation for clinical use in brachytherapy.
The data used to support the findings of this study are available from the corresponding author upon request.
S.J. Frank and K.S. Martirosyan are cofounders of C4 Imaging, LLC. S.J. Frank is an advisory board member and consultant for Varian Medical Systems, Inc. All other authors declare no conflicts of interest.
This work was supported in part by Cancer Center Support (Core) Grant CA016672 from the National Cancer Institute to MD Anderson Cancer Center and by the Prostate Cancer Foundation, the University Cancer Foundation via the Sister Institution Network Fund, the Institutional Research Grant program, and startup funds [to Steven J Frank] from The University of Texas MD Anderson Cancer Center. The authors thank Dr. Agatha Borne of the Departments of Veterinary Medicine & Surgery for his technical assistance. They also thank Christine F Wogan of MD Anderson’s Division of Radiation Oncology for editorial assistance.
Supplemental