Abstract
Objectives.
Mn-PyC3A is an experimental manganese- (Mn) based extracellular fluid MRI contrast agent that is being evaluated as a direct replacement for clinical gadolinium based contrast agents. The goals of this study were to use simultaneous PET-MR imaging to (1) compare the whole body pharmacokinetics, biodistribution, and elimination of Mn-PyC3A with the liver specific contrast agent Mn-DPDP, (2) determine the pharmacokinetics and fractional excretion of Mn-PyC3A in a rat model of renal impairment, and (3) to compare whole body elimination of Mn-PyC3A to gadoterate (Gd-DOTA) in a rat model of renal impairment.
Methods.
Mn-PyC3A and Mn-DPDP were radiolabeled with the positron emitting isotope Mn-52 via Mn2+ exchange with 52MnCl2. Dynamic simultaneous PET-MRI was used to measure whole body pharmacokinetics and biodistribution of Mn-52 immediately and out to 7 days following an intravenous 0.2 mmol/kg dose of [52Mn]Mn-PyC3A to normal or to 5/6 nephrectomy rats or a 0.01 mmol/kg dose of [52Mn]Mn-DPDP to normal rats. The fractional excretion, 1 day and 7 day biodistribution in rats after injection of 2.0 mmol/kg [52Mn]Mn-PyC3A (n=11 per time point) or Gd-DOTA (n=8 per time point) were quantified by gamma counting or Gd elemental analysis, respectively. Comparisons of Mn-PyC3A pharmacokinetics and in vivo biodistribution in normal and 5/6 nephrectomy rats and comparisons of ex vivo Mn vs. Gd biodistribution data in 5/6 nephrectomy were made with an unpaired t-test.
Results.
Dynamic PET-MR imaging data demonstrate that both [52Mn]Mn-PyC3A and [52Mn]Mn-DPDP are eliminated by mixed renal and hepatobiliary elimination, but that a greater fraction of [52Mn]Mn-PyC3A is eliminated by renal filtration. Whole body PET images show that Mn-52 from [52Mn]Mn-PyC3A is efficiently eliminated from the body while Mn-52 from [52Mn]Mn-DPDP is retained throughout the body. The blood elimination half-life of [52Mn]Mn-PyC3A in normal and 5/6 nephrectomy rats is 13 ± 3.5 min and 23 ± 12 min, respectively, P = 0.083. Area under the curve between 0 and 60 min post-injection (AUC0–60) in the bladder of normal and 5/6 nephrectomy rats are 2600 ± 1700 %ID/cc*min and 750 ± 180 %ID/cc*min, respectively, P = 0.024, while AUC0–60 in the liver of normal and 5/6 nephrectomy rats are 33 ± 13 and 71 ± 16, respectively, P = 0.011, indicating increased hepatobiliary elimination in 5/6 nephrectomy rats. The %ID of Mn from [52Mn]Mn-PyC3A and Gd from Gd-DOTA recovered from 5/6 nephrectomy rats 1 day after injection were 2.0 ± 1.1 and 1.3 ± 0.34, respectively, P = 0.10, and 7 days after injection were 0.14 ± 0.11 and 0.41 ± 0.24, respectively, P = 0.0041.
Conclusions.
Mn-PyC3A has different pharmacokinetics and is more efficiently eliminated than Mn-DPDP in normal rats. Mn-PyC3A is efficiently eliminated from both normal and 5/6 nephrectomy rats, with increased fractional hepatobiliary excretion from 5/6 nephrectomy rats. Mn-PyC3A is more completely eliminated than Gd-DOTA from 5/6 nephrectomy rats after 7 days.
INTRODUCTION
Gadolinium (Gd)-based contrast agents (GBCAs) for magnetic resonance imaging (MRI) play an integral role in modern radiology, but safety concerns over long-term Gd retention (1–6) and delayed onset toxicity (7–12) are driving research efforts to identify gadolinium-free alternatives for contrast enhanced MRI (13, 14). Gadolinium is associated with nephrogenic systemic fibrosis (NSF), a rare but debilitating condition observed in renally impaired patients that have received contrast enhanced MRI examinations (7, 15, 16). Through regulatory and market actions macrocylic GBCAs are now widely used and these are believed to be safer with respect to NSF risk. There are also safety concerns over long-term Gd retention and accumulation in the brain and body (1, 2, 17). This accumulation is observed in patients with normal renal function and intact blood brain barrier, is cumulative with repeat GBCA exposure, and has been observed following injection of both linear and macrocyclic GBCAs (17–20). These safety concerns are causing many referring physicians and radiologists to reconsider GBCA usage in renally impaired subjects, children, and those who need frequent contrast enhanced scans. However dose reduction or avoidance of GBCAs brings the risk of missed diagnosis or understaging.
Complexes of manganese (Mn) have been proposed as gadolinium-free alternatives to GBCAs and have received increasing attention (14). The Mn2+ ion is a strong T1-relaxation agent and is also an endogenously present nutritional element that can be eliminated by the human body (21, 22). There is precedence for Mn-based MRI contrast agents in clinical use. The manganese-based contrast agent mangafodipir or Mn-DPDP (Figure 1) was approved in the US and EU for liver imaging (23, 24), although the product is not currently marketed. A formulation of weakly chelated and bioavailable Mn called EVP-1001 (25) has completed Phase II clinical trials for imaging of myocardial viability (NCT00881075). A Mn-based nanoparticle formulation named SN132D (26) that is currently being developed for breast cancer imaging recently entered Phase I clinical trials (NCT04080024). An oral formulation containing manganese chloride has an approval for gastrointestinal imaging but is also no longer marketed, and an oral formulation containing manganese chloride with D-alanine and vitamin-B3 to stimulate enterohepatic Mn circulation is currently being evaluated in Phase III trials as a liver specific agent (NCT04119843).
Figure 1.
Chemical structures and corresponding pH 7.4 conditional stability constants (logKcond pH 7.4) of the liver specific manganese-based MRI contrast agent Mn-DPDP and experimental manganese-based MRI contrast agent Mn-PyC3A. The synthesis and stability constants of both complexes were previously reported (29, 32).
However, none of the Mn-based contrast agents that had marketing approvals or are under clinical development can serve as an interchangeable replacement for GBCAs. Most of these Mn-based contrast agents are designed for a single organ specific imaging indication and each has distinctly different pharmacokinetics than the extracellular fluid GBCAs that are used in the vast majority of contrast-enhanced examinations. For example, Mn-DPDP is effective for enhancing the hepatobiliary system but possesses distinctly different pharmacokinetics than the extracellular fluid GBCAs that are used in the vast majority of contrast-enhanced examinations, and thus cannot be used as a GBCA replacement. Mn-DPDP undergoes substantial Mn dissociation in blood plasma and a large fraction of the dissociated Mn2+ transiently accumulates in hepatocytes. Mn-DPDP must be infused to minimize the peak free Mn2+ concentration and avoid a vasodilatory effect caused by Mn2+ uptake in the heart muscle (27). On the other hand, extracellular fluid GBCAs are typically given as bolus injections, rapidly distribute through the blood and extracellular spaces, and are eliminated by renal filtration (28).
The complex Mn-PyC3A (Figure 1) was rationally designed as a direct replacement for extracellular fluid GBCAs (29). Mn-PyC3A has comparable relaxivity and pharmacokinetics to extracellular fluid GBCAs and has been shown to perform comparably to GBCAs like Gd-DTPA and Gd-DOTA for imaging blood vessels in baboons and imaging tumors in a mouse model of breast cancer, respectively (30, 31). Mn-PyC3A is roughly 100,000-fold more thermodynamically stable than Mn-DPDP at physiological pH and unlike Mn-DPDP, it is robust against Mn dissociation in vivo (29, 30, 32). The increased stability with respect to Mn2+ dissociation enables Mn-PyC3A to be administered as a bolus. Mn from injected Mn-PyC3A is also more efficiently eliminated from rats than Gd from injected Gd-DOTA (31), which is widely considered the most inert GBCA with respect to Gd deposition. Mn-PyC3A was also designed to be eliminated by mixed renal and hepatobiliary excretion (29), premised on the hypothesis that the hepatobiliary path will compensate for diminished renal function and enable Mn-PyC3A to be more efficiently eliminated from renally impaired patients than extracellular fluid GBCAs.
Manganese has an isotope, Mn-52, that has a 5.6 day half-life and decays by positron emission (33). The advent of simultaneous PET-MRI enables the assessment of whole body pharmacokinetics, biodistribution, and elimination of Mn-based contrast agent over a period of days. Here we used PET-MRI to first assess how [52Mn]Mn-PyC3A rational design alters the pharmacokinetics, Mn biodistribution, and elimination compared to the commercial contrast agent [52Mn]Mn-DPDP. We then used PET-MRI to quantify pharmacokinetics and elimination pathway of [52Mn]Mn-PyC3A in a rat model of renal impairment to determine if increased hepatobiliary elimination would compensate for decreased renal function. Finally, we compared the biodistribution and whole body elimination of injected Mn from Mn-PyC3A to that of Gd from injected Gd-DOTA in a rat model of renal impairment.
MATERIALS AND METHODS
Animal Model
All experiments were performed in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals, 8th Edition, and were approved by the institutional animal care and use committee of the institution where the work was performed. Normal Wistar rats and Wistar rats having undergone the 5/6 nephrectomy procedure in which in which one whole kidney and the upper and lower poles of the remaining kidney are excised were obtained from Charles River Labs (Wilmington, MA). All rats were purchased at 8–10 weeks of age.
A total of 52 rats were included in this study and were divided into five groups. Group 1 comprised n = 6 normal rats (3M/3F) receiving 0.2 mmol/kg [52Mn]Mn-PyC3A who underwent dynamic PET-MRI. Group 2 comprised n=6 5/6 nephrectomy rats (3M/3F) receiving 0.2 mmol/kg [52Mn]Mn-PyC3A who underwent dynamic PET-MRI. Group 3 comprised n=2 normal Wistar rats (1M/1F) receiving 0.01 mmol/kg [52Mn]Mn-DPDP who underwent dynamic PET-MRI. Group 4 comprised n=22 5/6 nephrectomy rats who received 2.0 mmol/kg Mn-PyC3A and were euthanized 1 day (6M/5F) or 7 days (5M/6F) post injection. Group 5 comprised n=16 5/6 nephrectomy rats who received 2.0 mmol/kg Gd-DOTA and were euthanized 1 day (4M/4F) or 7 days (3M/5F) post injection.
Contrast agents
Mn-PyC3A was prepared by using a previously reported protocol (29). Mn-DPDP was a gift from Dr. Alan Koretsky at the National Institute of Neurological Disorders and Stroke. Gd-DOTA was prepared from gadoterate meglumine solution (Guerbet). 52MnCl2 (t1/2 = 5.6 days) in 0.3 mL aqueous HCl was obtained from the Cyclotron Facility at the University of Alabama, Birmingham. A 0.50 M solution of [52Mn]Mn-PyC3A was prepared using a previously reported protocol (31). A 0.010 M solution of [52Mn]Mn-DPDP was prepared as follows. A 0.30 mL aliquot of 50 mM Mn-DPDP was spiked with 0.94 mCi 52MnCl2 in 0.030 mL aqueous HCl. The pH was carefully adjusted to pH 3.0 by addition of 1 M HCl and stirred at room temperature for 30 minutes. The pH was then adjusted back to pH 8.0 by addition of 1 M N-methyl-D-glucamine. The solution was then diluted with pure water to a total volume of 1.5 mL and filtered through a cation exchange cartridge (Supelco Discovery™ DSC-WCX). The final pH was pH 8.0. [52Mn]Mn-DPDP was the only 52Mn containing species detected by HPLC equipped with a gamma detector. The [52Mn]Mn-DPDP stock was sterile filtered into a sterile sealed glass vial.
PET-MR imaging
Rats were imaged in a 4.7 Tesla MRI scanner equipped with a PET insert (Bruker, Billerica, MA). Rats were anesthetized with isoflurane (4% for induction,1 to 1.5% for maintenance in medical air). After placement of a tail vein catheter for probe administration, rats were positioned prone on a custom-built cradle. Animals were kept warm using an air heater system and body temperature and respiration rate monitored by a physiological monitoring system (SA Instruments Inc., Stony Brook NY) throughout the imaging session. [52Mn]Mn-PyC3A was formulated at 0.5 M in sterile water and 0.4 μL per gram animal body weight was intravenously administered as a bolus via tail vein. [52Mn]Mn-DPDP was formulated at 0.01 M in sterile water and 1 μL per gram animal body weight was intravenously infused over 1 minute via tail vein. 4 – 11 MBq activity was administered to each rat.
Before contrast agent injection, T1-weighted MR images were acquired using a 3D Fast Low Angle Shot (FLASH) sequence with the following acquisition parameters: repetition time (TR)/echo time (TE) = 20 ms/3 ms, flip angle (FA) = 30°, field of view (FOV) = 80 × 65 mm2, matrix size = 267 × 200, 50 slices, slice thickness = 0.8 mm, and acquisition time = 3 min 20 sec). Immediately after contrast agent injection the FLASH sequence was repeated continuously with the PET acquisition performed simultaneously for 65 minutes. Rats were then returned to their cages. Rats were imaged again at 4 – 6 h, 3 – 4 d, and 7 d after injection for a period of of 30, 30, and 45 minutes, respectively.
PET-MR image analysis
Dynamic PET data was acquired in list-mode and were reconstructed using maximum likelihood expectation maximization (MLEM) algorithm with 12 iterations, 0.75 mm isotropic voxels, and binned into sequential time frames with durations of 10 × 20s, 7 × 60 s, 7 × 300 s, 2 × 600 s. For the later time point scans the entire 30 or 45 min PET data set was reconstructed into single images. Reconstructed PET data were analyzed using AMIDE software package (34). To study pharmacokinetics and biodistribution, volumes of interest (VOIs) over main organs including liver, heart, kidneys, and urinary bladder were defined on MR images and used for quantifying radioactivity for each PET frame. Results were expressed as percentage of injected dose per cubic centimeter of tissue (%ID/cc).
Time-activity curves were generated from VOIs in the heart, kidneys, liver, and urinary bladder out to 6 hours post-injection. Blood clearance was estimated by fitting the heart time-activity data to a biexponential model: %ID/cc(t) = Aexp(−αt) + Bexp(−βt), where %ID/cc(t) is %ID/cc at time t, α and β are the distribution and elimination rates, and A and B are the distribution and elimination coefficients, respectively. Distribution and elimination half-lives were calculated as ln(2)/α and ln(2)/β. Area under the time-activity curve (AUC0–60) was calculated by integrating the time-activity data recorded between 0 and 60 min.
Fractional excretion and ex vivo biodistribution in rats.
Rats were anaesthetized with isofluorane and administered 2.0 mmol/kg [52Mn]Mn-PyC3A (0.5 M in sterile water, 4 – 11 MBq) or Gd-DOTA (0.5 M in sterile water) via a 4 μL/g bolus injection into the tail vein. After contrast agent injection, the rats were housed in metabolic cages from the time of injection until euthanasia. Fractional excretion of [52Mn]Mn-PyC3A was estimated by collecting and quantifying radioactivity in all of the urine and feces excreted over the course of 1 day after [52Mn]Mn-PyC3A injection. The [52Mn]Mn-PyC3A and Gd-DOTA treated rats were euthanized via cardiac puncture 1 day or 7 days after injection. A total of 41 tissues were harvested for biodistribution studies from rats receiving [52Mn]Mn-PyC3A, and a subset of 23 tissues were taken from rats receiving Gd-DOTA. A full list of tissues analyzed is provided in Supplementary Tables S1 and S2. The contents of the stomach, cecum, intestines, and urinary bladder were emptied and also analyzed. Mn biodistribution was quantified by gamma counting and Gd biodistribution was quantified by elemental analysis by inductively coupled plasma mass spectrometry (ICP-MS).
Ex vivo biodistribution was also performed on the two [52Mn]Mn-DPDP dosed rats imaged by PET-MR at 8 days after the contrast agent injection. The [52Mn]Mn-DPDP dosed rats were returned immediately to metabolic cages after scans until the last scan.
Ex vivo Mn-52 quantification.
Ex vivo quantification of 52Mn activity in each injected dose, in the urine, and in the feces were determined using a Capintec CRC-15PET dose calibrator. Injected dose activity was determined from the difference in activity recorded in the loaded syringe and injection line prior to and after injection. Quantification of Mn-52 activity in ex vivo tissue samples was performed using a Perkin Elmer 2480 Wizard2 gamma counting system. Percentage injected (%ID) dose in ex vivo tissue samples was determined by comparing tissue counts to a measured aliquot of the injected dose. Bone, blood, fat, and muscle were estimated to account for 13%, 7%, 13%, and 40% of the total body weight. All measurements were corrected to account for decay. Limit of quantification (LOQ) was defined as 3 SD above the mean background counts per minute.
ICP-MS
Tissues were weighed and then digested overnight in concentrated nitric acid. Samples for elemental analysis were prepared by diluting an aliquot of the nitric acid digest into an aqueous solution of 0.1% Triton-X and 5% nitric acid. Gd quantification was performed using an Agilent 8800-QQQ ICP-MS system. A linear calibration curve ranging from 0.1 to 200 ppb Gd was generated daily using solutions prepared from NIST standard reference materials. A lutetium (Lu) internal standard was present during analysis and the calibration curve was generated by plotting the ratio of Gd:Lu counts vs. concentration of the calibration standards.
Statistical analysis.
Comparisons between Mn-PyC3A pharmacokinetics and biodistribution in normal and 5/6 nephrectomy rats and comparisons between Mn-PyC3A and Gd-DOTA biodistribution in 5/6 nephrectomy rats were performed using an unpaired t-test. P values less than 0.05 were considered statistically significant (GraphPad Prism 7, La Jolla, California).
RESULTS
Pharmacokinetics and in vivo biodistribution of [52Mn]Mn-PyC3A and [52Mn]Mn-DPDP in normal rats.
Dynamic and simultaneously recorded PET and T1-weighted MR images demonstrate rapid blood clearance and mixed renal hepatobiliary elimination of [52Mn]Mn-PyC3A and [52Mn]Mn-DPDP from normal rats. However, the two agents have different pharmacokinetics with a greater fraction of [52Mn]Mn-DPDP being eliminated via the hepatobiliary path than [52Mn]Mn-PyC3A. Figure 2A shows coronal maximum intensity projection (MIP) PET images of the torso recorded 1, 5, 10, 20, 40, and 60 min after injection of either contrast agent. The T1-weighted MR images were used to identify anatomical structures for PET data quantification (not shown). Time-activity curves of [52Mn]Mn-PyC3A and [52Mn]Mn-DPDP in the heart, kidney, liver, and bladder are shown in Figure 3 and pharmacokinetics parameters estimated from the time-activity data are shown in Table 1. Blood clearance pharmacokinetics were estimated from time-activity data recorded in the heart.
Figure 2.
(A) Maximum intensity projection (MIP) PET images of the entire torso of normal rats recorded at 1, 5, 10, 20, 40, and 60 min after injection of 0.2 mmol/kg [52Mn]Mn-PyC3A and 0.01 mmol/kg [52Mn]Mn-DPDP (4 – 11 MBq of Mn-52) demonstrate different in vivo biodistribution and different fractional renal:hepatobiliary excretion for the two contrast agents. Each PET frame is reconstructed over a duration of 180 seconds centered at the indicated time except the 1 min frame is reconstructed over 20 seconds. (B) Whole body MIP PET images of the normal rats recorded at 4 – 6 h, 3 or 4 d, and 7 d after injection of [52Mn]Mn-PyC3A and [52Mn]Mn-DPDP demonstrate a higher degree of whole body Mn-52 elimination following injection of [52Mn]Mn-PyC3A. Note the difference in scale for images recorded between 1–60 minutes, 4–6 hours, and ≥3 days after contrast agent injection.
Figure 3.
Representative time-activity curves recorded prior to and out to 6 hours following injection of 0.2 mmol/kg [52Mn]Mn-PyC3A (black circles) and 0.01 mmol/kg [52Mn]Mn-DPDP (red circles) in the heart, kidney, liver, and bladder of normal rats. Radiochemical dose of each agent was 4 – 11 MBq. The data highlight differences in [52Mn]Mn-PyC3A vs. [52Mn]Mn-DPDP pharmacokinetics such as greater fractional renal excretion of [52Mn]Mn-PyC3A and highly efficient [52Mn]Mn-PyC3A clearance. Note that Mn-52 activity in the heart and kidney decrease continuously following bolus injection of [52Mn]Mn-PyC3A, whereas Mn-52 activity initially decreases in the heart and kidney after [52Mn]Mn-DPDP injection but plateaus after about 35 min and 20 min, respectively.
Table 1.
Pharmacokinetics parameters estimated for PET time-activity curves recorded between 0 and 60 min after contrast agent injection.
| [52Mn]Mn-PyC3A in normal rats | [52Mn]Mn-DPDP in normal rats | [52Mn]Mn-PyC3A in 5/6 nephrectomy rats | P, [52Mn]Mn-PyC3A in normal vs 5/6 nephrectomy rats | |
|---|---|---|---|---|
| t1/2 distribution | 0.90 ± 0.53 min | 0.74 ± 0.50 min | 0.96 ± 0.89 min | 0.89 |
| t1/2 elimination | 13 ± 3.5 min | 6.2 ± 1.6 min | 23 ± 12 min | 0.0083 |
| AUC0–60 heart | 25 ± 8.5 %ID/cc*min | 24 ± 5.0 %ID/cc*min | 51 ± 9.4 %ID/cc*min | 0.0005 |
| AUC0–60 kidney | 240 ± 210 %ID/cc*min | 130 ± 19 %ID/cc*min | 320 ± 230 %ID/cc*min | 0.47 |
| AUC0–60 liver | 33 ± 13 %ID/cc*min | 140 ± 30 %ID/cc*min | 71 ± 16 %ID/cc*min | 0.0011 |
| AUC0–60 bladder | 2600 ± 1700 %ID/cc*min | 1200 ± 650 %ID/cc*min | 750 ± 180 %ID/cc*min | 0.024 |
[52Mn]Mn-PyC3A and [52Mn]Mn-DPDP each exhibit a biphasic elimination profile consistent with predominantly extracellular distribution, with elimination half-lives of 13 ± 3.5 min and 6.2 ± 1.6 min, respectively. MR signal enhancement and Mn-52 activity in the kidneys peak between 1 and 2 minutes after injection and rapidly decrease with concomitant bladder activity increase, consistent with urinary excretion. In the liver, MR signal enhancement and Mn-52 activity peak between 10 and 15 minutes after injection of either agent and then gradually decrease via biliary elimination evidenced by the concomitant accumulation of Mn-52 activity in the bowels. AUC0–60 in the bladder following injection of [52Mn]Mn-PyC3A and [52Mn]Mn-DPDP are 2600 ± 1700 %ID/cc*min and 1200 ± 650 %ID/cc*min, respectively, consistent with greater fractional urinary excretion of [52Mn]Mn-PyC3A, whereas AUC0–60 in the liver are 33 ± 13 %ID/cc*min and 140 ± 30 %ID/cc*min, respectively, consistent with greater fractional hepatobiliary excretion of [52Mn]Mn-DPDP. Dynamic PET imaging, Figure 2A, shows that a substantial amount of Mn-52 activity tracks to the bone and salivary gland within one hour of [52Mn]Mn-DPDP injection, but this distribution is not visible in the images collected after [52Mn]Mn-PyC3A injection. Figures 2A and 3 also show that Mn-52 is retained in the heart and kidney following [52Mn]Mn-DPDP injection, for example the time activity curves in these organs first decrease but reach a steady state plateau at roughly 35 minutes post injection in the heart and at 20 minutes post-injection in the kidneys consistent with retention. On the other hand [52Mn]Mn-PyC3A is very efficiently cleared from these tissues and the time activity curves in Figure 3 show a continuous decrease of activity with time consistent with elimination.
Mn administered as [52Mn]Mn-PyC3A is more efficiently eliminated than Mn administered as [52Mn]Mn-DPDP. Figure 2B shows 3D maximum intensity projection (MIP) PET images capturing the head, neck, and entire torso recorded at time intervals of 4 to 6 hours, 3 to 4 days, and 7 days after injection of [52Mn]Mn-PyC3A and [52Mn]Mn-DPDP. Within 4h of [52Mn]Mn-PyC3A injection, nearly all remaining injected Mn-52 activity is found within the excretory system with the majority of activity found within the bowel and urinary bladder. Residual activity was also observed in the stomach, presumably due to Mn-52 enterohepatic circulation. At 4h after [52Mn]Mn-DPDP injection, much of the remaining injected Mn-52 is also found in the excretory system, but high concentrations of Mn-52 remain in tissues including the kidneys, liver, brain, bone, heart, and salivary glands. We note that Mn-52 biodistribution observed by PET in the hours after [52Mn]Mn-DPDP injection is very similar to the PET biodistribution previously reported after intravenous injection of [52Mn]MnCl2 (33). Although small amounts of residual Mn-52 can be identified after injection of either contrast agent, the %ID remaining in each tissue is over an order of magnitude greater following injection of [52Mn]Mn-DPDP. Mn-52 levels decrease out to the 7 day time point after injection of both contrast agents, but the relative %ID retained following injection of [52Mn]Mn-DPDP vs [52Mn]Mn-PyC3A remain largely unchanged. The image for the [52Mn]Mn-DPDP injected rat at 7 days post injection shows substantial Mn present in the liver, bone, and salivary glands.
Ex vivo biodistribution of [52Mn]Mn-DPDP in normal rats.
Ex vivo biodistribution data collected 8 days after [52Mn]Mn-DPDP injection are summarized in Table 2. A total of 9.1 %ID and 6.1 %ID were recovered from all tissues analyzed from the male and female rat, respectively. The greatest %ID was found in the bone followed by the liver then by skin. A substantial portion of the injected Mn-52 was recovered from the skeletal muscle of the male rat, but residual activity in skeletal muscle was below the limit of quantitation for the female rat. For comparison, previously reported ex vivo biodistribution data collected 7 days after injection of 2.0 mmol/kg [52Mn]Mn-PyC3A to normal rats is included in Table 2. For each tissue analyzed, the %ID recovered following [52Mn]Mn-PyC3A injection is substantially lower than from [52Mn]Mn-DPDP injection.
Table 2.
Percentage of injected dose (%ID) in various organs recovered ex vivo 8 days after injection of 0.01 mmol/kg [52Mn]Mn-DPDP to normal rats or 7 days after injection of 2.0 mmol/kg [52Mn]Mn-PyC3A to normal rats (previously reported) (31). Only tissues where residual Mn-52 comprised > 0.05 %ID in at least one of the [52Mn]Mn-DPDP treated rats are shown, the complete ex vivo [52Mn]Mn-DPDP biodistribution data are shown in Figure S1.
| Mn biodistribution (%ID) | |||
|---|---|---|---|
| Mn-DPDP – 8 days | Mn-PyC3A – 7 daysa | ||
| Rat 1 - Female | Rat 2 - Male | ||
| Bone/Bone Marrow Sternum | 2.8 | 3.1 | 0.0158 |
| Brain | 0.051 | 0.072 | 0.0009 |
| Cecum | 0.11 | 0.092 | 0.0125 |
| Duodenum | 0.053 | 0.066 | <LOQ |
| Heart | 0.034 | 0.026 | <LOQ |
| Kidney | 0.24 | 0.43 | 0.0042 |
| Liver | 1.7 | 2.0 | 0.0181 |
| Pancreas | 0.25 | 0.29 | 0.0046 |
| Salivary Gland - Submandibular | 0.064 | 0.10 | 0.0013 |
| Seminal vesicle | N/A | 0.11 | 0.0028 |
| Skeletal muscle (rectus femoris) | <LOQ | 1.2 | <LOQ |
| Skin (base of tail) | 0.44 | 1.0 | <LOQ |
| Spinal cord (lumbar) | 0.10 | 0.040 | 0.0016 |
| Stomach | 0.10 | 0.11 | 0.0015 |
| Testes | 0.14 | 0.0004 | |
| Other tissues, see SI | 0.18 | 0.34 | 0.0051 |
| Total Tissue recovery | 6.2 | 9.1 | 0.058 |
[52Mn]Mn-PyC3A biodistribution was reported in a previous study (31).
Pharmacokinetics and in vivo biodistribution of [52Mn]Mn-PyC3A in 5/6 nephrectomy rats
The dynamic imaging data recorded prior to and out to 60 min after [52Mn]Mn-PyC3A injection to 5/6 nephrectomy rats demonstrates efficient contrast agent elimination, although at a slower rate and with different fractional excretion into the urine and feces compared to rats with normal renal function. Pharmacokinetic parameters following injection of 0.2 mmol/kg [52Mn]Mn-PyC3A in the heart, kidney, liver, and bladder of normal and 5/6 nephrectomy rats are compared in Table 1. The blood elimination half-life of [52Mn]Mn-PyC3A in 5/6 nephrectomy rats is 23 ± 13 min. AUC0–60 recorded in the heart of normal and 5/6 nephrectomy rats are 25 ± 8.5 %ID/cc*min and 51 ± 9.4 %ID/cc*min (P = 0.0005). AUC0–60 in the urinary bladder of 5/6 nephrectomy rats is 750 ± 180 %ID/cc*min, significantly less than that observed for [52Mn]Mn-PyC3A in normal rats (P = 0.024) and consistent with diminished renal function. Similar to [52Mn]Mn-PyC3A in normal rats, liver activity in 5/6 nephrectomy rats peaks between 10–15 minutes post-injection and then decreases due to biliary elimination. Liver AUC0–60 in the 5/6 nephrectomy rats is 71 ± 16, which is significantly greater than that observed in normal rats (P = 0.011) and consistent with increased fractional hepatobiliary elimination of for [52Mn]Mn-PyC3A in 5/6 nephrectomy rats.
Figure 4A compares coronal abdominal T1-weighted MR images recorded prior to and 5, 10, 20, 40, and 60 minutes after injection of 0.2 mmol/kg for [52Mn]Mn-PyC3A to normal and 5/6 nephrectomy rats; coronal abdominal PET images at the same time points are shown in Figure 4B. Both sets of images are consistent with increased hepatobiliary clearance in the 5/6NX rats.
Figure 4.
(A) Coronal abdominal T1-weighted MR images obtained prior to and 5, 10, 20, 40, and 60 min after injection of 0.2 mmol/kg [52Mn]Mn-PyC3A to normal rats and 5/6 nephrectomy rats demonstrate an increase in hepatobiliary excretion in 5/6 nephrectomy rats. The liver and kidneys are outlined in white and orange, respectively. (B) Coronal abdominal PET images obtained at 5, 10, 20, 40, and 60 min after injection of 0.2 mmol/kg [52Mn]Mn-PyC3A to normal rats and 5/6 nephrectomy rats. Liver and kidneys are outlined in white.
[52Mn]Mn-PyC3A is efficiently eliminated from 5/6 nephrectomy rats. MIP PET images comparing residual biodistribution in the head, neck, and torso of normal and 5/6 nephrectomy rats 1 and 7 days after [52Mn]Mn-PyC3A are shown in Figure 5. Similar to what is observed in normal rats, [52Mn]Mn-PyC3A is nearly entirely eliminated within 1 day after injection. The vast majority of activity remaining is located in the bowels.
Figure 5.
Coronal and sagittal MIP PET images capturing the head, neck, and entire torso of normal and 5/6 nephrectomy rats recorded at time intervals of 1 day and 7 days after injection of [52Mn]Mn-PyC3A demonstrate efficient whole body elimination in both normal and 5/6 nephrectomy rats.
Fractional excretion of [52Mn]Mn-PyC3A in 5/6 nephrectomy rats.
Quantification of 52Mn recovered in the urine and feces following an intravenously delivered dose of 2.0 mmol/kg in [52Mn]Mn-PyC3A demonstrate that the contrast agent is still efficiently eliminated from 5/6 nephrectomy rats. A total of 71 ± 4.8 %ID and 20 ± 4.4 %ID were recovered in the urine and feces 1 day after injection, totaling 91 ± 4.0 %ID and corresponding to 98% of the total recovered activity. Fractional excretion was 78 ± 4.7 % to the urine and 22 ± 4.7 % to the feces.
Ex vivo biodistribution and whole body elimination of [52Mn]Mn-PyC3A and Gd-DOTA in 5/6 nephrectomy rats.
Mn and Gd recovery in tissues recorded 1 day and 7 days after injection of 2.0 mmol/kg [52Mn]Mn-PyC3A or Gd-DOTA are compared in Table 3. A more extensive bio-distribution analysis was performed following [52Mn]Mn-PyC3A injection than after Gd-DOTA injection and the full results are available in Supplementary Table S1. At 1 day after injection, the total amount of 52Mn and Gd recovered from all tissues were 2.0 ± 1.1 %ID and 1.3 ± 0.34 %ID, respectively (P = 0.12). Following [52Mn]Mn-PyC3A injection, the majority (80%) of Mn-52 activity recovered from tissues was found in the gastrointestinal tract. The highest %ID were in the colon and cecum, together comprising 1.4 ± 1.0 %ID. An additional 0.20 ± 0.31 %ID was located in the remainder of the gastrointestinal tract.
Table 3.
Percentage of injected dose (%ID) in various organs and excreta 1 day and 7 days after injection of 4 – 11 MBq, 2.0 mmol/kg [52Mn]Mn-PyC3A or 2.0 mmol/kg Gd-DOTA to 5/6 nephrectomy rats and comparison of Gd vs Mn tissue concentration. Only tissues where both Mn and Gd were measured are shown. Additional tissues were analyzed for [52Mn]Mn-PyC3A and the complete biodistribution is shown in Supplementary Table S1. Data shown as mean ± SD, unpaired t-test. P values deemed statistically significant are denoted with an asterisk (*). LOQ = limit of quantification.
| 1 day biodistribution - %ID | 7 day biodistribution - %ID | |||||||
|---|---|---|---|---|---|---|---|---|
| Tissue or excreta analyzed | Mn | Gd | Gd:Mn | P | Mn | Gd | Gd:Mn | P |
| Adrenal Glands | 0.0002 ± 0.0004g | 0.0011 ± 0.0007f | 4.7 | 0.0012* | 0.0002 ± 0.0004g | 0.0008 ± 0.0005f | 3.5 | 0.0098* |
| Blood | 0.0002 ± 0.0007g | 0.026 ± 0.037d | 110 | 0.033* | 0.0001 ± 0.0002g | 0.014 ± 0.019f | 205 | 0.0272* |
| Bone/Bone Marrow (Sternum) | 0.085 ± 0.11g | 0.15 ± 0.064f | 1.7 | 0.16 | 0.035 ± 0.029g | 0.054 ± 0.036f | 1.5 | 0.2196 |
| Brain | 0.0040 ± 0.0010g | 0.0004 ± 0.0001f | 0.095 | <0.0001* | 0.0020 ± 0.0014g | 0.0002 ± 0.0001f | 0.075 | 0.0022* |
| Cecum | 0.55 ± 0.52g | 0.055 ± 0.024f | 0.10 | 0.015* | 0.0035 ± 0.0043g | 0.0049 ± 0.0017f | 1.4 | 0.3978 |
| Colon | 0.83 ± 0.68g | 0.045 ± 0.063f | 0.055 | 0.0050* | 0.017 ± 0.021g | 0.0035 ± 0.0034f | 0.20 | 0.0859 |
| Duodenum | 0.017 ± 0.0077g | 0.0045 ± 0.0024f | 0.26 | 0.0003* | 0.0011 ± 0.0011g | 0.0015 ± 0.0005f | 1.3 | 0.3536 |
| Eye | 0.0000 ± 0.0001g | 0.0009 ± 0.0002f | 21 | <0.0001* | <LOQg | 0.0004 ± 0.0004f | 0.0038* | |
| Heart | 0.0014 ± 0.0012g | 0.0024 ± 0.0005f | 1.8 | 0.048* | 0.0002 ± 0.0002g | 0.0010 ± 0.0006f | 5.0 | 0.0007* |
| Ileum | 0.031 ± 0.022g | 0.015 ± 0.02f | 0.48 | 0.12 | 0.0006 ± 0.0009g | 0.0035 ± 0.0030f | 5.7 | 0.0072* |
| Jejunum | 0.040 ± 0.025g | 0.0094 ± 0.0037f | 0.23 | 0.0031* | 0.0008 ± 0.0007g | 0.0023 ± 0.0008f | 2.9 | 0.0004* |
| Kidney | 0.051 ± 0.026g | 0.26 ± 0.069f | 5.2 | <0.0001* | 0.0017 ± 0.0013g | 0.13 ± 0.042f | 79 | <0.0001* |
| Liver | 0.078 ± 0.022g | 0.17 ± 0.073f | 2.1 | 0.0012* | 0.043 ± 0.014g | 0.039 ± 0.029f | 0.91 | 0.6956 |
| Lung w/ bronchi | 0.0013 ± 0.0011g | 0.0080 ± 0.0023f | 6.3 | < 0.0001* | 0.0002 ± 0.0002g | 0.0022 ± 0.0011f | 10 | <0.0001* |
| Ovary | <LOQc | 0.0050 ± 0.0024b | <0.0001* | 0.0001 ± 0.0001d | 0.0017 ± 0.0010c | 27 | 0.0034* | |
| Pancreas | 0.0081 ± 0.0045g | 0.010 ± 0.0024f | 1.2 | 0.30 | 0.0018 ± 0.0011g | 0.0036 ± 0.0032f | 2.0 | 0.0990 |
| Skeletal muscle (rectus femoris) | 0.036 ± 0.098g | 0.31 ± 0.25f | 8.6 | 0.0039* | 0.0095 ± 0.013g | 0.10 ± 0.11e | 11 | 0.0096* |
| Skin (base of tail) | 0.089 ± 0.17g | 0.22 ± 0.084f | 2.5 | 0.051 | <LOQg | 0.042 ± 0.023f | <0.0001* | |
| Spleen | 0.0009 ± 0.0015g | 0.013 ± 0.0034f | 14 | <0.0001* | 0.0003 ± 0.0003g | 0.0040 ± 0.0017f | 14 | <0.0001* |
| Stomach | 0.11 ± 0.29g | 0.0083 ± 0.0052f | 0.076 | 0.37 | 0.0028 ± 0.0015g | 0.0015 ± 0.0009f | 0.53 | 0.0442* |
| Testes | 0.0061 ± 0.0044d | 0.010 ± 0.0074b | 1.6 | 0.30 | 0.0020 ± 0.0006c | 0.0037 ± 0.0004b | 1.8 | 0.0042* |
| Thymus/ Parathyroid gland | 0.0003 ± 0.0006g | 0.0036 ± 0.0007f | 11 | <0.0001* | 0.0001 ± 0.0001g | 0.0012 ± 0.0007f | 16 | <0.0001* |
| Uterus + vagina | <LOQc | 0.0081 ± 0.0028b | 0.0003* | 0.0001 ± 0.0002d | 0.0035 ± 0.0020c | 25 | 0.0024* | |
| Other tissues, see Table S1 | 0.043 ± 0.084g | 0.020 ± 0.045g | ||||||
| Total tissue recovery | 2.0 ± 1.1g | 1.3 ± 0.34f | 0.66 | 0.12 | 0.14 ± 0.11g | 0.41 ± 0.24f | 2.9 | 0.0043* |
n = 3,
n = 4,
n = 5,
n = 6,
n = 7,
n = 8,
n=11.
At 7 days after injection, there was >3-fold more Gd retained in 5/6 nephrectomy rats compared to Mn from injected Mn-PyC3A. The total amount of Mn-52 and Gd recovered from all tissues were 0.14 ± 0.11 %ID and 0.41 ± 0.24 %ID, respectively (P = 0.0043). On day 7, the Gd tissue concentration was either significantly greater than or not significantly different than exogenous Mn concentrations in nearly every tissue. Gd tissue concentrations were significantly higher in the adrenal glands, blood, eye, heart, ileum, jejunum, kidney, lung, ovary, skeletal muscle, skin, spleen, stomach, testes, thymus, and uterus, whereas the only tissue where residual exogenously administered Mn levels were significantly higher than Gd was the brain. The largest percentage of Mn is recovered in the liver, bone and colon, each corresponding to < 0.05 %ID. In all other tissues analyzed, Mn concentrations were < 0.01 %ID.
DISCUSSION
In this study we used quantitative positron emission tomography combined with MRI to study the whole body pharmacokinetics and biodistribution of two Mn-based contrast agents in rat models. The Mn-52 isotope is ideally suited to this task because of its long 5.6 day half-life and its decay which occurs exclusively through positron emission. The lability of the Mn2+ makes radiolabeling Mn-based contrast agents straightforward with complete incorporation of label and no need for HPLC purification. Extracting Mn concentration from MR images is challenging because the contrast agent is detected indirectly by its effect on tissue water hydrogen atoms. Measuring T1 change post Mn injection provides some insight but to determine concentration one requires the relaxivity of the agent and this depends on the chemical form (chelated, free ion, protein bound) and its microscopic distribution (e.g. slow water exchange between tissue compartments may limit the relaxation effect) (21, 35). Moreover MRI is limited in its ability to detect low levels of contrast agent because of the background signal due to native T1. PET has exceptional sensitivity and the signal is directly proportional to the isotope concentration and does not depend on chemical form. As a result it is possible to quantify blood and organ pharmacokinetics and also detect the distribution of residual amounts of the injected dose. Because there is no complex background in PET, it was obvious to detect Mn accumulation in tissues like the salivary gland and bone with PET. A major unresolved question with GBCAs is how much of the injected Gd is retained in the human body and where it is distributed. For Mn-based contrast agents, this could be addressed in the days after dosing by a combined Mn-52 PET-MRI study. Gadolinium lacks a suitable positron emitting isotope for such studies although recent work has demonstrated how radiolabeling GBCAs with the pseudolanthanide yttrium-86 positron emitting isotope can be used to accurately approximate residual Gd biodistribution (36).
The marked differences in whole body pharmacokinetics and distribution of Mn-52 observed after injection of [52Mn]Mn-PyC3A compared to [52Mn]Mn-DPDP underscore how the pharmacokinetics and biodistribution of intravenously injected Mn depends strongly upon how the Mn is chelated. The PyC3A chelator was designed to bind Mn2+ tightly and to be kinetically inert with respect to Mn2+ release. At pH 7.4, Mn-PyC3A is >100,000-fold more stable than Mn-DPDP at pH 7.4 (29, 32). The observation that Mn-52 administered as [52Mn]Mn-PyC3A is more efficiently eliminated than Mn-52 administered as [52Mn]Mn-DPDP is consistent with expected behavior and with prior studies that evaluated Mn release in vivo following injection of either complex. For example, in a human pharmacokinetic study with Mn-DPDP, it was determined that 80% of the administered Mn is released from the DPDP ligand in the body, consistent with a chelate that is kinetically labile in vivo (37). On the other hand, in a baboon pharmacokinetic study, tandem high pressure liquid chromatography (HPLC) – ICP-MS analysis of blood plasma serially collected prior to and out to 60 min after injection of Mn-PyC3A indicated that Mn-PyC3A is robust against Mn release and did not uncover any major biotransformation products, consistent with a chelate that is thermodynamically stable and kinetically inert in vivo (30). The PET-MR imaging data shown here cannot distinguish between chelated and dissociated Mn-52, but it does demonstrate how a substantially greater percentage of the injected 52Mn tracks to tissues known to accumulate Mn (brain, salivary gland, heart, bone) when injected as [52Mn]Mn-DPDP, consistent with higher concentrations of free Mn2+ released following injection of [52Mn]Mn-DPDP.
PET-MR imaging of [52Mn]Mn-PyC3A in normal and 5/6 nephrectomy rats demonstrates that [52Mn]Mn-PyC3A is efficiently eliminated even in the presence of renal insufficiency. Dynamic PET-MR imaging data recorded prior to and out to 60 min after injection of [52Mn]Mn-PyC3A rats shows that renal insufficiency does alter Mn-PyC3A both the elimination rate and elimination path. There is increased exposure to the compound in the 5/6 nephrectomy rats, but the data also demonstrate how hepatobiliary elimination provides a compensatory excretion path in these animals. Delayed imaging recorded 1 and 7 days after [52Mn]Mn-PyC3A injection show near complete whole body elimination similar to that observed in normal rats, with no areas of marked accumulation. Although fractional elimination in rodents does not necessarily predict that which will occur in humans, the data does demonstrate how hepatobiliary elimination can compensate for diminished renal filtration to efficiently eliminate contrast agents that were designed for partial hepatobiliary elimination.
The efficiency with which [52Mn]Mn-PyC3A is eliminated is further evidenced by the observation that Mn-52 from [52Mn]Mn-PyC3A is more completely eliminated from 5/6 nephrectomy rats than is Gd from an equal molar dose of Gd-DOTA. Gd-DOTA is widely considered to be the most inert GBCA with respect to Gd release. This result in 5/6 nephrectomy rats mirrors a prior study where Mn-PyC3A was shown to be more efficiently eliminated than Gd-DOTA from normal rats (31).
There are a few limitations to the study. The PET-MR imaging and ex vivo biodistribution studies quantify concentrations of Mn-52 and Gd, but cannot distinguish what chemical form residual Mn-52 or Gd exists in. Another limitation is that although our data demonstrate that hepatobiliary elimination of Mn-PyC3A increases in the presence of renal insufficiency in rats, it remains unknown as to whether this result will translate to humans. Fractional excretion profiles in rodents and humans can differ vastly. For example, the GBCA Gd-BOPTA exhibits roughly 50% hepatobiliary elimination in rats but only 2–4% of the GBCA is eliminated by the hepatobiliary path in humans (38). Another limitation is that the experiments to perform PET-MR imaging and ex vivo fractional excretion and biodistribution were performed using different doses of [52Mn]Mn-PyC3A. The 0.2 mmol/kg dose [52Mn]Mn-PyC3A dose for imaging studies was chosen to maximize signal enhancement in T1-weighted images. We reasoned that 0.2 mmol/kg dose would be high enough to provide a strong T1 shortening effect but not so high that signal is lost due to T2* effects. The 2.0 mmol/kg [52Mn]Mn-PyC3A dose administered in the fractional excretion and ex vivo biodistribution experiment was chosen to remain consistent with a previously reported biodistribution analysis in normal rats (31). Although in vivo PET-MR imaging and ex vivo biodistribution data are both consistent with efficient whole body elimination of [52Mn]Mn-PyC3A from 5/6 nephrectomy rats, dynamic PET-MR imaging data after a 0.2 mmol/kg dose of [52Mn]Mn-PyC3A showed > 2-fold higher hepatobiliary elimination than in normal rats, whereas quantification of Mn-52 in urine and feces from 5/6 nephrectomy rats receiving a 2.0 mmol/kg dose of [52Mn]Mn-PyC3A showed a 50% increase in hepatobiliary elimination compared to normal rats receiving an equal dose. One explanation for this discrepancy is saturation of biliary elimination at higher doses, although this needs to be investigated further. Saturable biliary clearance has been previously reported for Gd-BOPTA (39). Similarly, [52Mn]Mn-PyC3A and [52Mn]Mn-DPDP pharmacokinetics and biodistribution were studied using unequal doses reflecting anticipated and actual clinical doses, respectively. Mn-DPDP dissociates upon injection to the high relaxivity free Mn2+ ion which is avidly taken up by hepatocytes delivering a sufficiently high concentration to the liver to shorten T1 and increase signal even at this relatively low dose. Mn-PyC3A remains intact and acts as an extracellular contrast agent. This requires a higher dose that is comparable to the GBCAs.
In conclusion, Mn-PyC3A is more efficiently eliminated from normal rats than the clinical Mn-based contrast agent Mn-DPDP and is also efficiently eliminated from 5/6 nephrectomy rats and with increased fractional hepatobiliary excretion. Mn-PyC3A is more efficiently eliminated from 5/6 nephrectomy rats than an equal dose of Gd-DOTA.
Supplementary Material
Acknowledgements
We would like to thank Dr. Suzy Lapi and her research group at the University of Alabama, Birmingham for providing us with 52Mn isotope for our PET-MR and ex vivo biodistribution studies. We would like to Dr. Alan Koretsky and his research group at the National Institute of Neurological Disorders and Stroke for providing us with Mn-DPDP. We also thank Alana Ross at the Martinos Center for Biomedical Imaging, who helped organize and execute the ex vivo biodistribution studies.
E. Sources of Support: This work was supported by grants from the National Institutes of Health (K25HL128899, R44DK113906, R01DK120663, R44CA239935, S10OD010650, S10OD023503, and S1OD025234) and the Department of Energy (DESC0015773).
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