Characterization of IRDye 800CW
UV/vis and Fluorescence (1× PBS) measurements yielded an absorption max. of 774 nm, an emission max. of 789 nm, and an extinction coefficient 240,000. Purity by HPLC at 780 nm was >98%.
Mass spectrometry results were as follows: m/z [M+H+] calc. 1003.2, found 1,003.3, 100%; [M+2H+] calc. 520.1, found 502.1, 45%; [M+Na+], calc. 1,025.3, found 1,025.2, 16%. The MS results are fully consistent with the expected structure of the IRDye 800CW carboxylate dye.
1H NMR results were as follows: (labels refer to Fig. 1a) A/A′ δ 7.71/7.74 (d, J = 1.5 Hz, 2H); C/C′ δ 7.74/7.76 (dd, J = 1.5, 8.1/8.4 Hz, 2H); D/D′ δ 7.21/7.26 (d, J = 8.3/8.4 Hz, 2H); K/K′ δ 7.77/7.82 (d, J = 14/7/14.5 Hz, 2H); J/J′ δ 6.07/6.13 (d, J = 14.3/14.0 Hz, 2H); Q δ 7.25 (d, J = 8.8 Hz, 2H); R δ 7.85 (d, J = 8.8 Hz, 2H); T/Z δ 3.97/3.97 (t, J = 6.9 Hz, 4H); CC δ 2.97 (t, J = 7.2 Hz, 2H); M/M′ δ 2.65 (m, J = 5.7 Hz, 4H); X δ 2.17 (t, J = 7.5 Hz, 2H); N δ 1.96 (m, J = 5.6 Hz, 2H); AA/BB δ 1.85/1.82 (m/m, 4H); U δ 1.73 (tt, J = 7.5 Hz, 2H); W δ 1.59 (tt, J = 7.5 Hz, 2H); V δ 1.36 (tt, J = 7.7 Hz, 2H); I/I′ δ 1.26/1.25 (s, 12H).
The 1H NMR assignments were developed from the 1D, COSY, HSQC, and HMBC spectra using standard pulse sequences for those experiments.
Dosing Solution Analysis
Dosing solutions were analyzed to verify the amount of material delivered to animals (Table 2). ICG was difficult to dissolve in the saline vehicle. The instructions for the preparation of ICG indicated that the lyophilized product should be dissolved in sterile water, which was supplied by the manufacturer. To facilitate solubility, samples were placed in an ultrasonicator and repeatedly forced through a syringe needle to aid solubility prior to administration. The amount of ICG administered varied from 116.9% to 132.4% of the intended dose at 20 mg/kg in the safety study. Animals in the pharmacokinetic study received 95.5% of the intended dose of 5 mg/kg. Homogeneity of the dosing solutions had a coefficient of variation of 2.3–13.4% for the 20 mg/kg solutions and 5.1% for the 5 mg/kg dosing solution.
In contrast, solubility of IRDye 800CW in saline was good even at the 20-mg/kg dose level. The amount and variation in the amount of ICG or IRDye 800CW at the different dose levels is shown in Table 2.
The method for recovery of dye in plasma and tissue was validated using spiked samples. Saline or plasma was spiked with 50.0 μg/mL of ICG. For IRDye 800CW, plasma was spiked with either 0.5 or 50.0 μg/mL of dye. The percent recovery of ICG from saline was 103.9%, while recovery from plasma was 78.1% based on triplicate analyses. After samples were frozen for 1 day, recovery of ICG was 98.9% for saline and 80.4% from plasma. With IRDye 800CW, plasma recovery was 62.7% and 90.9% at 0.5 and 50 μg/mL, respectively. When samples were frozen for 2 days, recovery was 60.8% at 0.5 μg/mL and 82.0% at 50 μg/mL.
To test recovery from tissue, liver was spiked with IRDye 800CW (100 μg/1.0–1.5 g), homogenized, and extracted. Recovery from liver was 56.9%. Analyses were done in triplicate.
Animals were assigned to treatment groups according to weight in order to avoid differences between groups. No statistical significance between initial or final body weights was observed between treatment groups, indicating that no overt toxicity occurred. No toxic effects were observed among any of the animals in this study during routine daily observations. For some animals that received either indocyanine green or IRDye 800CW following intravenous injection, a green color persisted around the injection sites for several days. For animals that received intradermal injections of IRDye 800CW on the dorsal surface of the feet, a green color persisted for several days on all animals. No test or control article-related toxicity was observed at the administration sites following histopathologic evaluation, which correlated with clinical observations.
Tissue distribution was determined in male and female rats given 5 mg/kg of IRDye 800CW. Tissues and plasma were obtained 1 h after IV administration of IRDye 800CW or 2 h after ID administration and processed as described in “Materials and Methods”. Overall means were similar for males and females and between routes of administration for lung, liver, spleen, and muscle (Fig. 2a, b). Slightly higher levels of IRDye 800CW were seen in lung, spleen, and muscle of males compared to females. Liver values were lower after ID administration compared to IV administration. The highest tissue levels were seen in kidney after IV administration, with higher levels found in males compared to females. The reverse was true for ID administration. Levels of IRDye 800CW were greater in ovaries than in testes, and the amount of IRDye 800CW in ovaries was greater in females after ID administration compared to IV administration. No IRDye 800CW was detected in testes 2 h after ID administration. IRDye 800CW was not detected in brain following either IV or ID administration.
Uptake of IRDye 800CW in the popliteal lymph nodes varied considerably with the route of administration (Fig. 2c, d). IRDye 800CW levels were much greater after ID administration compared to the IV route. Of all tissues, popliteal uptake was greatest when normalized per gram of tissue after ID administration compared to other organs or tissues.
In addition to tissue distribution, the pharmacokinetics of plasma levels of both ICG and IRDye 800CW were performed. Pharmacokinetic analysis of IV ICG was performed to serve as a control as published pharmacokinetic values were available for comparison. A single dose level of 5 mg/kg was used for both IV and ID administration. ICG was only administered IV, whereas IRDye 800CW was administered both by the intravenous and intradermal routes. Multiple injections were made in both rear feet for all ID injections in this study.
Pharmacokinetic parameters were determined for IRDye 800CW and ICG using non-compartmental pharmacokinetics. Results of the clearance for IRDye 800CW following IV and ID administration of 5 mg/kg are shown in Fig. 3a, c. The time to peak plasma concentrations was fairly consistent among animals after IV administration, but differed considerably after ID administration. Average values were determined by combining the values obtained for individual animals. The different times to reach peak plasma concentrations after ID administration may be a result of differences in the release of IRDye 800CW. The terminal half-life of ICG determined in this study (Fig. 3b), 6.2 min after IV administration of 5 mg/kg, is similar to that reported in the literature for humans and other species [15, 16]. Clearance half-life of IRDye 800CW following IV injection was determined to be 35.7 min, whereas the half-life following ID injection was 236.5 min. Although urine levels of IRDye 800CW were not evaluated, green-colored urine was observed in the bladders of animals in the tissue distribution and pharmacokinetic studies, which indicates this was a potential route of excretion. Excretion by the kidneys is also indicated by the high tissue levels of IRDye 800CW in kidneys of animals in the tissue distribution study.
Blood samples were collected from all animals in the safety study just prior to euthanasia. When samples were clotted or processing was delayed, unreliable values were obtained that were not used to determine group responses. Group averages for hematology data are summarized in Table 3, while data for individual animals are shown in Electronic Supplementary Material (ESM) Fig. 1a–p. After statistical analyses were performed on the data, no clinically significant dose-related changes in hematology were observed 24 h or 14 days after exposure to the test or control articles for either males or females.
For males, all parameters analyzed were generally within the normal ranges for the respective tests (Table 3; ESM Fig. 1a–p). A slight elevation was noted in the hematocrit and hemoglobin of control males and high-dose IV IRDye 800CW groups, but were deemed insignificant. While slight differences were noted between some of the values for day 15 as compared to day 2, the values fell within the normal ranges. There was no evidence of a dose or route of administration-related response.
Values were similar for males and females with the exception of white blood cells, which were slightly lower in females. There was no evidence of a dose-related difference between intravenous or intradermal administration.
Liver and Kidney Function
For males analyzed 24 h post-injection, all parameters were within the normal range for the respective tests (Table 4; ESM Fig. 2a–m), with the exception of LDH and creatine kinase that were elevated outside the normal range in animals that received the vehicle control of intravenous saline. Alkaline phosphatase was elevated above the normal range in all animals regardless of treatment.
At 15 days post-injection, all parameters analyzed for male liver function were within the normal ranges for the respective tests (Table 4; ESM Fig. 2a–m), with the exception of GGT in the mid-dose IRDye 800CW group and creatinine kinase in the high-dose IV IRDye 800CW group that were elevated outside the normal range. Examination of the data for individual animals (ESM Fig. 2c, f) indicated that the averages for these values were skewed by a single animal. The average value for LDH (Table 4; ESM Fig 2d) was similarly skewed in the ID IRDye 800CW group by two animals. All of the individual skewed values were from different animals. None of these animals had more than a single value outside of the normal range. Globulin was also elevated in the mid-dose IRDye 800CW group, and total bilirubin was elevated in the ICG group. As with the 24-h groups, alkaline phosphatase levels were elevated above the normal range in all animals regardless of treatment, except for the animals in the ID IRDye 800CW group which was in the normal range. There was no evidence of a dose-related response or from the two different routes of administration. Overall, hepatic function test results were similar in the 14-day and 24-h treatment animals. The elevated values described above did not have any clinical significance for the treatment groups 15 days after exposure to the test or control article.
Similarly, females showed no evidence of a dose-related response or differences from the two different routes of administration 24 h post-injection (Table 4; ESM Fig. 2a–m). Values obtained from females were similar to those seen in males, with the exception of ALT and alkaline phosphatase which were lower in females than in males. As seen in males, LDH and creatinine kinase were elevated outside the normal range in animals that received the saline vehicle control intravenously. LDH was also elevated above the high normal range in animals that received ICG. ALT values were below normal in animals that received the 5- and 20-mg/kg doses of IRDye 800CW, but no clinical significance could be attributed to this observation.
Again, at 15 days post-injection, there was no evidence of a dose-related response or differences from the two different routes of administration for liver function in females. Values obtained from females were similar to those seen in males, with the exception of alkaline phosphatase which was not elevated in females compared to males. As seen in males, LDH was slightly elevated outside the normal range in females that received the saline vehicle control intravenously. LDH was also slightly elevated after 14 days in the vehicle control, ICG, and low-dose IRDye 800CW groups. Creatine kinase was elevated in the vehicle control group. Globulin levels were also elevated above the high normal range in animals that received mid and high doses of IRDye 800CW. ALT values were below normal in animals that received the 5- and 20-mg/kg doses of IRDye 800CW, but no clinical significance could be attributed to this observation. Results from females 14 days after treatment were similar to those from females in the 24-h treatment group.
At 24 h post-injection, all parameters analyzed were within the normal ranges for the respective tests for males and females (Table 5). There was no evidence of either a dose or route of administration-related response. Although potassium and phosphorous levels were lower in females than males, the difference was not clinically significant.
Fifteen days post-injection, all parameters analyzed were within the normal ranges for the respective tests (Table 5) for males and females, with the exception of the male mid-dose IRDye 800CW group potassium levels in which the mean was slightly lower than the low normal value. There was no evidence of either a dose or route of administration-related response.
Gross necropsies were conducted on all animals in the safety study, including extra animals in the high-dose groups. Because no lesions were observed in the extra animals, no tissues were submitted for histopathologic evaluation. Some animals in the highest group appeared to have excess red fluid (presumably blood) around the brain, so additional rats were included in the day 2 female high-dose groups, which were dosed separately from the day 2 males. No consistent findings were observed to verify the observations in males, and there was no confirmation of adverse findings after histopathologic evaluation of the brains of males or females in the day 2 or day 15 groups, nor was this seen in animals in the tissue distribution study that had 1- or 2-h exposures to the test article.
Organs were weighed at necropsy to determine any gross effects of the test or control article on specific organ systems. No statistically significant differences were observed between groups when absolute organ weights were compared or when relative organ weights were compared. One male rat in the day 15 ID IRDye 800CW group had an enlarged spleen. On histopathologic evaluation of the spleen from this animal, increased hemopoiesis (grade 1) was noted. As grade 1 hemopoiesis was also seen in animals in the vehicle control group and animals in other groups that received test article by IV administration, the observation of an enlarged spleen in this animal was deemed to be unrelated to test article administration.