Patent Application
20180271470
Type 1 diabetes mellitus caused by the autoimmune destruction of insulin producing pancreatic beta cells affects approximately 0.2% of the world's population. Pancreatic islet transplantation combined with immune suppression has been shown to temporarily allow for partial or full insulin independence in patients with type 1 diabetes mellitus, but no permanent treatment yet exists. An imaging modality capable of monitoring the decline of functional beta cell mass and the viability of islet or stem-cell derived beta cell transplants would be invaluable to future therapeutic investigations.
Human pancreatic islets occupy approximately 4.5% of the pancreas volume, and are composed of a mixture of β, α, γ, δ, and ε cells. As individual islets vary in size from 25 to 400 μm in diameter and are non-uniformly distributed throughout the pancreas, quantification is challenging using noninvasive anatomical imaging techniques such as magnetic resonance imaging (MRI) or computed tomography (CT). Alternatively, positron emission tomography (PET) is a technique that typically involves quantifying the in vivo distribution of a biologically relevant moiety via tracking with a positron emitting radioisotope. Compared with MRI and CT, PET has significantly greater imaging sensitivity and inherently probes physiology rather than anatomy, which may prove useful in the clinical quantification of functional beta cell mass.
Mn2+, in a behavior that mimics Ca2+, is freely transported through voltage dependent Ca2+ channels (VDCCs). Because VDCC activation is required for the release of insulin from beta cells, Mn2+ has been proposed as a molecular imaging agent for probing beta cell function and mass using manganese-enhanced magnetic resonance imaging (“MEMRI”). However, this technique is limited by the significant cellular toxicity of Mn2+ at the concentration necessary for increasing image contrast, and slow biological clearance of manganese, which prevents the possibility of repeated contrast administration.
The present disclosure addresses the aforementioned drawbacks by providing a method for quantitatively imaging pancreatic beta cells using positron emission tomography (“PET”). Radiomanganese is administered to a subject. The radiomanganese can be free isotopes or can be linked or otherwise conjugated to other molecules. Data are acquired from a region-of-interest containing a pancreas of the subject using a PET system. An image of the region-of-interest is reconstructed from the acquired data. The reconstructed image depicts a preferential uptake of the radiomanganese in pancreatic beta cells in the subject. The image is processed with a computer system to estimate a quantitative parameter of one of pancreatic beta cell mass or pancreatic beta cell function.
It is another aspect of the disclosure to provide a method for imaging pancreatic beta cells using PET. A first image and a second image of a subject acquired with a PET system following administration of radiomanganese to the subject are provided to a computer system. The first image depicts a first radiomanganese activity in the subject and the second image depicts a second radiomanganese activity in the subject. A difference between the first activity and the second activity is computed and a pancreatic beta cell mass, such as a functional beta cell mass, is quantified based on the computed difference.
It is another aspect of the disclosure to provide a method for assessing a pancreatic tissue transplant using PET. Radiomanganese is administered to a subject who has received a pancreatic tissue transplant, which may be a stem cell-derived tissue transplant. An image of the subject is acquired with a PET system and a pancreatic beta cell mass is computed from the acquired image. A report is then generated based on the computed pancreatic beta cell mass. The report contains information associated with an assessment of transplant viability of the pancreatic tissue transplant.
It is yet another aspect of the disclosure that a pharmacological agent can be administered to the subject before administering radiomanganese, wherein the pharmacological agent modulates the uptake of divalent metals by pancreatic beta cells. The pharmacological agent can inhibit or stimulate divalent metal uptake.
The foregoing and other aspects and advantages of the present disclosure will appear from the following description. In the description, reference is made to the accompanying drawings that form a part hereof, and in which there is shown by way of illustration a preferred embodiment. This embodiment does not necessarily represent the full scope of the invention, however, and reference is therefore made to the claims and herein for interpreting the scope of the invention.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Described here are methods for imaging beta cells in pancreatic tissue using radioisotopes of manganese, which may be referred to as radiomanganese. Example radioisotopes of manganese include Mn-52g, Mn-52m, and Mn-51. As one example, radiomanganese can be used to image pancreatic beta cells, in which radiomanganese shows a preferential uptake. This provides for applications such as quantifying beta cell mass (e.g., functional beta cell mass), assessing transplant viability, monitoring the efficacy of drug treatments, and so on.
Radiomanganese is advantageous for positron emission tomography (“PET”) imaging applications because of its favorable physical and chemical properties. As an example of its favorable chemical properties, radiomanganese can be rapidly and stably chelated by 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, which is also known as DOTA. This chelation enables bioconjugate applications of radiomanganese, including conjugating radiomanganese with antibodies, peptides, small molecules, whole cells, and so on. As another example of its favorable chemical properties, manganese can also be integrated into manganese-based nanoparticles, such as manganese-oxide nanopartides, manganese containing porphysomes, and so on.
The physical properties of radiomanganese enable different uses and applications based on the different isotopes used to form the radiomanganese. Table 1 illustrates some of the physical properties of the Mn-51, Mn-52m, and Mn-52g radioisotopes of manganese.
Mn-52g has a long half-life relative to the other isotopes of manganese. This longer half-life makes Mn-52g less suitable for clinical use, but more advantageous for preclinical studies. For instance, the longer half-life means that Mn-52g radiomanganese can be shipped worldwide. Mn-52m has a relatively short half-life and produces unfavorable gammas. Although Mn-52m radiomanganese could be used for clinical use, it is more difficult to produce and handle than Mn-51. Mn-51 has a short half-life, a strong positron branch, and no prominent gammas. These properties make Mn-51 advantageous for clinical use.
Thus, in general, based on the physical properties of the different isotopes of manganese, it is contemplated that radiomanganese containing Mn-52g will be advantageous for preclinical studies, whereas radiomanganese containing Mn-51 will be more advantageous for clinical studies and use.
Another beneficial property of manganese is that it behaves biologically like Ca2+, which enables its use in applications such as neural tract tracing, pancreatic beta cell imaging, measuring cardiac efflux rates for determining myocardial infarction, insulinoma imaging, and so on.
Radiomanganese also has the benefit of being a magnetic resonance imaging (“MRI”) contrast agent that shortens the longitudinal relaxation time (“TI”) of nuclear spins proximate the contrast agent. As a result of this property, radiomanganese can be advantageous for imaging studies that implement simultaneous PET/MRI.
An example of an existing manganese-based MRI contrast agent is Mangafodipir. A significant drawback to manganese-based MRI contrast agents is that these agents are cytotoxic, especially at the large amount of contrast agent that is necessary to achieve a suitable increase in image contrast. The slow excretion of manganese from contrast agents such as Mangafodipir also means that repeated imaging of a subject is not practical.
Because MnCl2 has the same final biodistribution as Mangafodipir, radiomanganese containing MnCl2 may provide complimentary diagnostic value in both PET imaging and MRI. As an added benefit, using radiomanganese containing MnCl2 provides reduced toxicity to the subject as compared to the higher toxicity Mangafodipir. For instance, radiomanganese can provide a suitable image contrast at much lower doses than are required for manganese-based MRI contrast agents, such as Mangafodipir. As an example, radiomanganese can be administered with doses in a sub micromolar range, such as a picomolar (“pM”) range, while still producing appreciable image contrast. The rapid blood clearance of radiomanganese containing MnCl2 also enables rapid scanning.
As mentioned above, radiomanganese can be used for quantifying beta cell mass, such as by quantifying functional beta cell mass. The noninvasive determination of functional beta cell mass is very useful for monitoring the progression of type-1 and type-2 diabetes, as well as other applications including monitoring the viability of transplanted insulin-producing cells. Previous work implementing manganese-enhanced MRI has shown promise for beta cell mass determination through voltage-dependent calcium channel (“VDCC”) internalization of Mn2+. However, these methods require the injection of bulk cytotoxic manganese contrast agent, which limits the clinical utility of such methods.
In addition to imaging pancreatic beta cells and measuring properties associated with those cells, radiomanganese can be used for other applications, including immunoPET applications, DMT-1 reporter gene imaging, neural tract tracing, PET/MRI applications with radiomanganese-based nanoparticles, insulinoma imaging, porphysome imaging, cell tracking, and small molecule studies. These additional imaging applications can be used for imaging the pancreas or other tissues and organ systems, whether or not in connection with studying the pancreas. As one example, radiomanganese can be used in immunoPET applications by conjugating the radiomanganese with molecules used in immunotherapy treatments. The radiomanganese can be imaged with PET to monitor the efficacy of the immunotherapy. These applications may have specific utility for predinical studies.
The methods described in the present disclosure make use of radiomanganese and PET imaging to image the pancreas and to monitor the physical, chemical, physiological, or other properties of pancreatic beta cells. For instance, radiomanganese can be used to quantify beta cell mass, monitor pancreatic function, assess the viability of transplanted insulin-producing cells, evaluate the efficacy of drugs or other treatment schemes, and so on.
Referring now to
The images acquired from the subject following administration of radiomanganese are then analyzed to quantify the desired properties of pancreas, such as beta cell mass or beta cell functions, as indicated at step 108. Methods for processing the image are described below.
In some applications, a pharmacological agent can be administered to the subject to modulate divalent metal (and thus radiomanganese) uptake in pancreatic beta cells. Techniques such as these can be useful in beta cell mass (BCM) quantification studies for the subtraction of non-specific exocrine pancreas uptake by stimulation or blocking (e.g., through glibendamide or nifedipine) of beta cell VDCCs following baseline imaging. On the other hand, the pulsatile nature of calcium transport may increase test-retest variability for bolus injection techniques. This effect could possibly be mitigated by administering 51MnCl2 as an intravenous infusion over 5-15 minutes.
Referring now to
Radiomanganese is then administered to the subject, as indicated at step 204. The radiomanganese can be administered as free isotopes of radiomanganese, or the radiomanganese can be linked to other molecules, such as by conjugation with an antibody or a peptide. Images are then obtained from the subject following administration of the radiomanganese, as indicated at step 206. While these images are preferably PET images, images can also be obtained with MRI, as indicated at step 208. In some applications, images can be acquired with an integrated PET/MRI system, which allows for simultaneous, or near-simultaneous, acquisition of both PET images and magnetic resonance images.
The images acquired from the subject following administration of radiomanganese are then analyzed to quantify the desired properties of pancreas, such as beta cell mass or beta cell functions, as indicated at step 210. Methods for processing the image are described below. For example, images with and without pharmacological modulation of radiomanganese uptake can be compared to quantify pancreatic beta cell function or mass. These comparisons may include computing the difference between two such images, correlating activities in one image with another, and so on. In some implementations, a report can be generated based on this comparison. For instance, the report may include numerical data indicating one or more quantitative values. As another example, the report may include an image, such as a digital image computed as a difference between two images. In this latter example, the report includes an image matrix having pixel values associated with difference values, which may be associated with a quantification of pancreatic beta cell function or mass.
Referring now to
The imaging hardware system 302 generally includes a PET scanner having a radiation detector ring assembly 310 that is centered about the bore 312 of the PET scanner. The bore 312 of the PET scanner is sized to receive a subject 314 for examination. Prior to imaging, the subject 314 is administered a radioisotope, such as a radionuclide or radiotracer. As described in the present disclosure, the subject can be administered radiomanganese or a radiotracer or other agent containing radiomanganese. The subject may also be administered other pharmacological agents before imaging, such as pharmacological agents that modulate beta cell activity by suppressing or stimulating divalent metal uptake.
Positrons are emitted by the radiomanganese as it undergoes radioactive decay. These positrons travel a short distance before encountering electrons at which time the positron and electron annihilate. The positron-electron annihilation event 316 generates two photons that travel in opposite directions along a generally straight line 318.
The radiation detector ring assembly 310 is formed of multiple radiation detectors 320. By way of example, each radiation detector 320 may include one or more scintillators and one or more photodetectors. Examples of photodetectors that may be used in the radiation detectors 320 include photomultiplier tubes (“PMTs”) or avalanche photodiodes (“APDs”). The radiation detectors 320 are thus configured to produce a signal responsive to the photons generated by annihilation events 316. The signal responsive to the detection of a photon is communicated to a set of acquisition circuits 322. The acquisition circuits 322 receive the photon detection signals and produce signals that indicate the coordinates of each detected photon, the total energy associated with each detected photon, and the time at which each photon was detected. These data signals are sent the data acquisition system 304 where they are processed to identify detected photons that correspond to an annihilation event 316.
The data acquisition system 304 generally includes a coincidence processing unit 324 and a sorter 326. The coincidence processing unit 324 periodically samples the data signals produced by the acquisition circuits 322. The coincidence processing unit 324 assembles the information about each detected annihilation event 316 into a set of numbers that indicate when the event took place and the position in which the event was detected. This event data is then processed by the coincidence processing unit 324 to determine if any two detected photons correspond to a valid coincidence event.
As one example, the coincidence processing unit 324 may determine if any two detected photons are in coincidence as follows. First, the times at which two photons were detected should be within a predetermined time window, for example, within 0-12 nanoseconds of each other, such as within a time window of 3-4 nanoseconds. Second, the locations at which the two photons were detected should lie on a line 318 that passes through the field of view in the PET scanner bore 312. Each valid coincidence event represents the line 318 connecting the two radiation detectors 320 along which the annihilation event 316 occurred, which is referred to as a line-of-response (“LOR”). The data corresponding to each identified valid coincidence event is stored as coincidence data, which represents the near-simultaneous detection of photons generated by an annihilation event 316 and detected by a pair of radiation detectors 320.
The coincidence data are communicated to a sorter 326 where the coincidence events are grouped into projection images, which may be referred to as sinograms. The sorter 326 sorts each sinogram by the angle of each view, which may be measured as the angle, θ, of the line-of-response 318 from a reference direction that lies in the plane of the detector ring assembly 302. For three-dimensional images, the sorter 326 may also sort the sinograms by the tilt of each view. The sorter 326 may also process and sort additional data corresponding to detected photons, including the time at which the photons were detected and their respective energies.
After sorting, the sinograms are provided to the data processing system 306 for processing and image reconstruction. The data processing system 306 may include a data store 328 for storing the raw sinogram data. Before image reconstruction, the sinograms may undergo preprocessing to correct for random and scatter coincidence events, attenuation effects, and other sources of error. The stored sinogram data may thus be processed by a processor 330 located on the data processing system 306, by the operator workstation 308, or by a networked workstation 332.
The operator workstation 308 typically includes a display 334; one or more input devices 336, such as a keyboard and mouse; and a processor 338. The processor 338 may include a commercially available programmable machine running a commercially available operating system. The operator workstation 308 provides the operator interface that enables scan prescriptions to be entered into the PET system 300. In general, the operator workstation 308 may be in communication with a gantry controller 340 to control the positioning of the detector ring assembly 310 with respect to the subject 314 and may also be in communication with the data acquisition system 304 to control operation of the imaging hardware system 302 and data acquisition system 304 itself.
The operator workstation 308 may be connected to the data acquisition system 304 and data processing system 306 via a communication system 342, which may include any suitable network connection, whether wired, wireless, or a combination of both. As an example, the communication system 342 may include both proprietary or dedicated networks, as well as open networks, such as the internet.
The PET system 300 may also include one or more networked workstations 332. By way of example, a networked workstation 332 may include a display 344; one or more input devices 346, such as a keyboard and mouse; and a processor 348. The networked workstation 332 may be located within the same facility as the operator workstation 308, or in a different facility, such as a different healthcare institution or clinic.
The networked workstation 332, whether within the same facility or in a different facility as the operator workstation 308, may gain remote access to the data processing system 306 or data store 328 via the communication system 342. Accordingly, multiple networked workstations 332 may have access to the data processing system 306 and the data store 328. In this manner, sinogram data, reconstructed images, or other data may be exchanged between the data processing system 306 or the data store 328 and the networked workstations 332, such that the data or images may be remotely processed by a networked workstation 332. This data may be exchanged in any suitable format, such as in accordance with the transmission control protocol (“TCP”), the internet protocol (“IP”), or other known or suitable protocols.
In this example, the in vivo biodistribution of 52Mn2+ was investigated noninvasively with PET imaging. This example study also assessed the feasibility of using Mn-PET to probe beta cell mass and function.
Mn-52 was produced as described by S. A. Graves, et al., in “Novel Preparation Methods of 52Mn for ImmunoPET Imaging,” Bioconjug. Chem., 2015; 26:2118-2124. Aliquots of the buffered 52Mn solution (0.01M NaOAc, pH 6.5) were diluted to the desired injection volume (˜200 μl) with phosphate-buffered saline (PBS), typically 2-4 MBq (50-100 μCi) per subject.
Two strains of mice, ICR (Envigo, Indianapolis, Ind.) and C57BL/6J (The Jackson Laboratory, a Harbor, Me.), were employed for in vivo imaging studies. All mice were approximately ten weeks of age at the time of the experiments. Pre-diabetic C57BL/6J ob/ob mice carrying the homozygous obese spontaneous leptin mutation were obtained from the Jackson Laboratory. Mice had access to food and water ad libitum, except under fasting condition when access to food was restricted for 6-12 hours. All animal experiments were performed under the approval of the University of Wisconsin Institutional Animal Care and Use Committee.
The acquisition of PET images was performed in the Inveon μPET/μCT scanner (Siemens Preclinical Solutions, Knoxville, Tenn.). To study the long-term biodistribution and clearance of 52Mn2+ in the mouse body, approximately 3.7 MBq (100 μCi) of 52Mn2+ was injected intravenously (IV) in female ICR mice. Due to the long physical half-life of 52Mn (t1/2: 5.6 d), PET scans were recorded at multiple time-points between 1 hour and 13 days post injection of the radiotracer. Before each scan, mice were anesthetized with isoflurane (4% induction; 1% maintenance) and placed in the scanner in prone position. 30×106-40×106 coincidence events per mouse static PET scans were acquired (time window, 3.432 ns; energy window, 350-650 keV) and the PET images were reconstructed in Inveon Acquisition Workplace (Siemens Preclinical Solutions, Knoxville, Tenn.) workstation using a non-scatter-corrected three-dimensional Ordered Subset Expectation Optimization/Maximum a Posteriori (OSEM3D/MAP) algorithm. Region-of-interest (ROI) analysis was performed after organs were manually delineated on the PET images. Tissue uptake values were reported as standardized uptake value (SUV), which is normalized to whole body radiomanganese concentration.
To acquire dynamic PET scans, mice were anesthetized with isoflurane and the lateral tail vein was catheterized. Simultaneous with the administration of approximately 1.7 MBq (50 μCi) of 52Mn2+ as a fast IV bolus, one-hour scans were recorded and list-mode files were binned into 46 frames (12×5 s, 6×10 s, 6×30 s, 10×60 s, 6×150 s, 6×300 s) and the images reconstructed using a non-scatter-corrected OSEM3D/MAP algorithm. In another study, 1.7 MBq (50 μCi) of 52Mn2 was continuously infused with a syringe pump (Kd Scientific, Model 780100) over a period of 30 min, starting at the beginning of the PET scan. List-mode files were framed into 30×2 min frames and reconstructed using the above-mentioned algorithm.
To investigate the specificity of 52Mn2+ for beta cells, a series of studies were performed where 52Mn2+ uptake was manipulated through the pharmacologic stimulation or inhibition of the insulin secretory pathway. In these experiments, 0.74-1.85 MBq (20-50 μCi) of 52Mn2+ was administered IV into either female ICR or C57BL/6J mice and static PET scans were acquired at one hour post injection. During tracer administration, mice were awake. Mice were only anaesthetized by isoflurane (1%) immediately before PET imaging.
Ex vivo biodistribution studies were performed in all groups of mice in order to validate the results of PET imaging and obtain a more complete biodistribution profile of 52Mn2+. Following the last imaging time point, mice were euthanized by C02 asphyxiation and the organs of interest were removed, wet-weighed, and counted on an automated gamma-counter (Wizard 2480, Perkin Elmer). The tissue uptake of 52Mn2+ was reported as SUV (mean±SD).
Mouse pancreatic islets were isolated by collagenase digestion. Briefly, mice were placed under shallow anesthesia and sacrificed via cervical dislocation. The mouse common bile duct was cannulized and 3-5 mL of an ice-cold solution containing type XI collagenase (0.5 mg/mL; Sigma Aldrich, St. Louis, Mo.) and bovine serum albumin (BSA; 0.2 mg/mL; Sigma Aldrich, St. Louis, Mo.) in Hank's balanced salt solution (HBSS; Invitrogen, Carlsbad, Calif.) was injected into the mouse pancreas. After inflation, the pancreas was removed, placed in a glass vial containing 5 mL of the collagenase solution, and incubated in a shaking water bath at 37° C. Samples were digested for approximately 30 min after which the digests were centrifuged at 50×g for 2 min and islet pellets were washed three times with 30 mL of an ice-cold BSA (0.2 mg/mL) in HBSS solution. The pellet was resuspended and islets handpicked into 35 mm petri dishes. Following isolation, islets were placed in RPMI1640 media supplemented with penicillin (100 U/mL; Invitrogen), streptomycin (100 μg/mL; Invitrogen), and FBS [10% (wt/vol); Sigma] and incubated overnight at 37° C. in a 5% CO2 atmosphere.
The uptake of 52Mn2+ by murine islets of Langerhans was determined under several stimulatory or inhibitory conditions. Batches of 50 islets were transferred into 0.45 μm filtered bottom 1 mL centrifuge vials (Thermo Fisher Scientific, Grand Island, N.Y.), 500 μL of Krebs-Ringer buffer (KRB: 118 mM NaCl, 5.4 mM KCl, 2.4 mM CaCl, 1.2 mM MgSO4, 1 mM KH2PO3, 20 mM HEPES; pH 7.4) containing a low glucose concentration (1 mM D-glucose) were added and the islets incubated for 30 min at 37° C. After removing the supernatant following centrifugation at 50×g for 5 min, 250 μL of KRB containing glucose, diazoxide (50 μM; Tocris Biosciences, Bristol, UK), or tolbutamide (250 μM; Selleckchem, Houston, Tex.) were added, and the vials were spiked with 370 kBq (10 μCi) of 52Mn2+. After 15 min of incubation, the solutions were filtered and islets were washed three times with KRB. The 52Mn2+ radioactivity in the islet pellets were measured in an automated gamma counter (Perkin Elmer).
To corroborate the correlation between 52Mn2+ pancreatic uptake and the mechanism of insulin release in beta cells, the insulin secretory pathways were stimulated in vivo using glucose and glibendamide (Tocris Biosciences, Bristol, UK), which promotes insulin release in beta cells via blockade of ATP-sensitive potassium channel (KATP). Mice were injected intraperitoneally (IP) with 100 μL of glucose (1 g/kg) or glucose (1 g/kg)+glibenclamide (5 mg/kg) in PBS, 15 min before the IV injection of 0.74-1.85 MBq (20-50 μCi)52Mn2+. Static PET scans were acquired one hour after the injection of the radiotracer, after which ex vivo biodistribution was performed.
VDCC blockade was achieved in vivo via a 20 mg/kg IP injection of 3,5-dimethyl 2,6-dimethyl-4-(2-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (nifedipine; MP Biomedicals, LLC, Santa Ana, Calif.) in dimethyl sulfoxide. To activate KATP channels, mice received an IP injection of a 20 mg/kg dose in PBS of 7-Chloro-3-methyl-4H-1,2,4-benzothiadiazine 1,1-dioxide (diazoxide; Tocris Biosciences, Bristol, UK), a clinically used potent KATP agonist Both groups of mice were injected with 0.74 MBq (20 μCi) of 52Mn2+, 15 min after the administration of either nifedipine of diazoxide. Whole-body PET scans were acquired at one hour post injection, after which ex vivo biodistribution was performed.
Type 1 diabetes was induced in female ICR mice via a single IP injection of 180 mg/kg streptozotocin (STZ; MP Biomedical, LLC, Santa Ana, Calif.), a toxin that selectively destroys pancreatic beta cell. The injectable STZ solution (12.5 mg/mL) was prepared freshly in phosphate buffered saline (PBS). The weight of each mouse was measured daily, and blood glucose levels were recorded every other day with a glucometer (TRUEresult, Trividia Health Inc., Fort Lauderdale, Fla.) using blood samples collected from the tail vein. Mice were considered diabetic after two consecutive blood glucose readings above 250 mg/dL and were used for PET imaging studies one week after the injection of STZ. To evaluate 52Mn2+ pancreatic uptake on diabetic mice, 0.74 MBq (20 μCi) of activity were administered IV and static PET images recorded one hour after administration of the tracer. Ex vivo biodistribution was carried out following PET acquisition.
Given the altered glucose metabolism in obese mice, we compared the pancreatic uptake of 52Mn2+ in lean (wild-type) and obese (ob/ob) mice on the C57BL/6J background. For PET imaging, 0.74 MBq (20 μCi) of 52Mn2+ was IV injected into obese mice or lean controls one hour prior to PET scan acquisition. Subsequent 52Mn2+ accumulation in the pancreas and other organs of interest was quantified.
A minimum sample size of three (n=3) was used in all in vitro and in vivo experiments. The uptake of 52Mn2+ in the different tissues was reported as SUV (mean±SD) and the differences between groups were evaluated for significance using a two-tailed Student's t-test. Differences were considered statistically significant at P<0.05.
Production yields of up to 5.92 MBq/μAh (355 MBq/h @ 60 ρA) were achieved using a natCr pellet pressed into a silver disc substrate. Mn-52 was eluted in <1 mL of 0.01 M NaOAc buffer (pH˜6.5) from a ˜150 mg AG 1×8 column which had been previously rinsed with ethanol. Thin layer chromatographs confirmed the Mn(II) oxidation state following elution. End of bombardment radionuclidic purity was measured to be >99.5% by efficiency-calibrated high-purity germanium (HPGe) gamma spectrometry measurements. The only radionudidic impurity observed was <0.5% Mn-54 (t1/2=312.1 d), which does not decay by positron emission.
The in vivo biodistribution of 52Mn2+ was investigated noninvasively with PET and ex vivo gamma counting.
Because the whole-body distribution of 52Mn2+ occurred largely within the first hour after IV administration (
As is shown in
Previous reports have shown that most volatile anesthetics, including isoflurane, impair insulin secretion by inhibiting the deactivation of KATP channels. Plausibly, the observed decrease in pancreatic uptake of 52Mn2+ resulted from mice being anesthetized through the full extent of the studies during the dynamic PET scans. To investigate the impact of isoflurane anesthesia on pancreatic uptake, one hour post injection biodistribution experiments were performed under different administration conditions in either anesthetized (1% isoflurane) or awake mice. Isoflurane significantly inhibited the accumulation of 52Mn2+ in the pancreas regardless of the administration regime (e.g., rapid bolus, continuous infusion) or glucose stimulation, suggesting that isoflurane indeed acts as an indirect VDCC inhibitor via the upstream inhibition of KATP channel closure.
To corroborate the mechanism of 52Mn2+ uptake in the pancreas and its specificity for beta cells, an in vitro 52Mn2+ uptake study was performed in islets isolated from obese (ob/ob) mice. Due to the similarities between Mn2+ and Ca2+ ions, Mn2+ uptake by beta cells occurs via influx through VDCC (
To verify the specificity of 52Mn2+ accretion in the pancreas noninvasively using PET imaging, 52Mn2+ pancreatic uptake was pharmacologically manipulated in vivo through the inhibition or stimulation of insulin secretion. In line with the in vitro results, inhibition of VDCC by direct blockade with nifedipine, or activation of KATP with diazoxide resulted in a significant (P<0.0001) abrogation of the PET signal within the pancreas.
The correlation between pancreatic 52Mn2+ uptake and beta cell functional was investigated in a murine model of type-1 diabetes. Diabetes was induced in ICR female mice via injection of a single STZ (180 mg/kg) dose, a well-established and extensively studied model in the literature. As observed in
Imaging studies were also performed in C57BL/6J mice carrying the lepob (ob/ob) spontaneous mutation that result in animal obesity and pre-diabetic syndrome (i.e. pre-type 2 diabetes), which is another model that is well-established and extensively studied in the literature. Increased 52Mn2+ accumulation in the pancreas of pre-type 2 diabetic ob/ob mice was observed in the PET data (
The loss, dysfunction, or both, of pancreatic beta cells is a significant component of both type 1 and type 2 diabetes. The ability of beta cells to sequester divalent metal ions (e.g. Ca2+, Mn2+, Zn2+, and Co2+) is an important aspect of the production and release of insulin. While beta cell function has been widely investigated based on the measurement of Ca2+ currents in vitro, little progress has been achieved in exploring beta cell divalent metal intake for measurement of function in vivo. Part of this is due to the lack of effective methods to track the distribution of most of these metals in vivo. Fortunately, Mn2+ has both magnetic and nuclear properties that facilitate its noninvasive detection by both MRI and PET. In the example study discussed above, the use of radioactive manganese for noninvasive PET imaging of the mouse pancreas was investigated. When injected intravenously, 52Mn2+ accumulated largely into pancreatic tissue with exceptionally fast blood extraction kinetics. 52Mn2+ uptake in the pancreas, which peaked within minutes following administration in healthy mice, was on the order of 5-6 SUV with slow clearance half-life of approximately 8 days.
Two isotopes of manganese present themselves as excellent candidates for manganese-based PET: Mn-52 (t1/2: 5.6 d, β+: 29.6%, Eβave: 0.24 MeV) and Mn-51 (t1/2: 45.6 min, β+: 97.1%, Eβave: 0.96 MeV). Due to its high energy gamma emissions and relatively long half-life, Mn-52 is well-suited for preclinical small animal research. Mn-51, on the other hand, has excellent decay properties that are more amenable to clinical applications. With its longer half-life, Mn-52 can be shipped nationally or internationally, whereas Mn-51 must be produced and utilized on-site.
One of the more common production strategies for Mn-52 involves the low energy (Ep+<20 MeV) proton irradiation of natCr metal targets. In this example study, an ethanol/HCl-based anion exchange chromatography that allows for a Mn-52 recovery efficiency of greater than 60% in less than 2 hours was implemented. Overall, contamination including heavy metals such as natCr (<0.1 μg) was very low, making the isolated Mn-52 suitable for preclinical investigations.
The results of the example study described above revealed that the observed pancreatic radioactivity was a result of the specific uptake of 52Mn2+ by pancreatic beta cells. By pharmacologically manipulating the mechanism of insulin secretion in isolated islets in vitro, the uptake of radioactive 52Mn2+ could be correlated with Ca2+ uptake. This agrees with other in vitro/ex vivo studies using non-radioactive Mn2+ that reported a correlation between Mn2+ uptake and beta cell functional capacity. Similarly, in vivo studies demonstrated that the chemical inhibition of insulin release, using nifedipine or diazoxide, resulted in a decline in the accumulation of 52Mn2+ within the pancreas. In addition, the stimulation of insulin secretion with glibenclamide (5 mg/kg) led to an increase in 52Mn2+ uptake in the pancreas. Overall, the results of this example study indicated that the 52Mn2+ uptake observed in the pancreas was largely mediated by and dependent on the functional beta cell mass.
The example study also investigated whether PET imaging with radiomanganese was sensitive enough to detect changes in beta cell mass that occur during diabetes progression and disease. First, an experimental animal model of STZ-induced type-1 diabetes, which showed a ˜60% reduction in the pancreatic accumulation of 52Mn2+ while uptake in other organs remained unaltered, was employed. This indicates the ability of radiomanganese PET imaging for revealing extreme cases of beta cell loss. The gradual loss of beta cell mass under chronic exposure to low doses of STZ could be examined using Mn-51 (t1/2=46 min) for longitudinal PET imaging.
In the obese mouse (ob/ob) model of pre-type 2 diabetes, significant (P<0.0001) enhancement in pancreatic uptake of 52Mn2+ was observed. These results agree with previous reports showing that ob/ob mice do not progress to type 2 diabetes, but rather present markedly higher beta cell mass compared with lean mice at 10 weeks of age. An increased beta cell mass in this population is consistent with the increased pancreatic uptake of 52Mn2+ that was observed in this example study, which demonstrates the potential of radiomanganese PET for noninvasively quantifying variations in beta cell mass within the context of type 2 diabetes, particularly at the early stage of disease progression.
Because there can be significant changes in beta cell function long before changes in beta cell mass are observed, it is advantageous that the relative uptake of radiomanganese in vivo depends on the functional beta cell mass. This ability has the potential to shed light on the survival of beta cell transplants, and on the rate of functional beta cell mass decline in asymptomatic type 1 diabetic patients.
The diffusion of Mn2+ through beta cell VDCCs depends on functional activation by glucose or drug-based stimulation. This has been shown in this example study through in vitro and in vivo functional suppression and enhancement studies. For a given state of glucose activation, the amount of Mn2+ cellular internalization is relative to the number of available VDCCs, which should be proportional to functional beta cell mass. Furthermore, accurate quantification of functional beta cell mass can include subtraction of nonspecific exocrine pancreas tracer uptake of the radiotracer. This may be accomplished through pharmacological manipulation to acutely suppress beta cell uptake using nifedipine, isoflurane, diazoxide, or other suppressing agents.
Our studies also provided valuable information on the kinetics of the whole-body distribution of 52Mn2+. Besides specifically accreting in pancreatic islets, 52Mn2+ also accumulated in the liver, heart, kidneys, and salivary gland. Despite the significant kidney uptake, no clear sign of renal excretion was observed and most of the 52Mn2+ clearance occurred through the hepatobiliary system. From the analysis of the image-derived dynamic TAC of the heart, a 52Mn2+ circulation half-life of 10.7±3.5 s following a rapid bolus injection was estimated, which aligned with previous studies showing an extremely fast (˜0.8 min) blood clearance of Mn-54 in dogs. Such fast extraction kinetics and the lack of evidence of 52Mn2+ metabolites in blood indicate that compartmental modeling could be used to describe 52Mn2+ uptake in a more quantitative manner.
Several PET tracers have been studied as potential beta cell imaging agents. [11C]-Dihydrotetrabenazine (DTBZ) and the 18F-labeled DTBZ analog FP-(+)-DTBZ have been shown to have high affinity for the type 2 vesicular acetylcholine transporter (VMAT2), which is expressed on the surface of rodent beta cells, but appears to be entirely absent from pancreatic exocrine tissue. Unfortunately, primate models have shown very low pancreatic uptake of [11C]-DTBZ, which suggests that the degree of VMAT2 expression is species-dependent.
Another category of tracers includes radiolabeled (e.g. F-18, Ga-68, Cu-64, In-111) derivatives of exendin-4, a glucagon-like protein-1 receptor (GLP-1R) agonist. Fluorescence microscopy has shown that GLP-1R is only located on beta cells within the human pancreas, making it an attractive molecular imaging target due to its specificity. Unfortunately, exendin-4 also suffers from low pancreatic uptake (less than 0.3% ID/g in Sprague-Dawley rats at 1 h post injection whereas proximal kidney uptake was greater than ˜25% ID/g). In general, the implementation of tracers targeting surface receptors of beta cells is challenging, considering the low total mass of beta cells, diffuse pancreatic distribution, and heterogeneous receptor expression.
Aside from following the decline in beta cell mass of type 1 diabetic patients, there is a pressing need for a noninvasive method for longitudinal imaging of pancreatic islet transplants. Islet transplantation has been shown to lead to insulin independence for several years in patients with type 1 diabetes, but has not been widely adopted due to the need for donor tissues. Recent advances in selective stem cell differentiation techniques will likely lead to wider clinical adoption of beta cell transplantation therapies. The ability to noninvasively track the survival and function of transplanted beta cells would enable research into the patient-specific efficacy of immune-modulating therapies and the development of new therapeutic strategies. The use of radiomanganese PET for monitoring islet transplant survival is compelling, since it can be performed repeatedly over time.
In this example study, methods for the production of Mn-51 on low-energy medical cyclotrons were investigated. In general, Mn-51 was produced by proton irradiation of electrodeposited isotopically-enriched Fe-54 targets followed by anion exchange chromatography. Initial 51MnCl2 pharmacokinetic characterization in mice and predicted human dosimetry showed promise for a variety of PET applications, including VDCC activation imaging in pancreatic beta cells.
All reagents used in this example study were obtained from commercial vendors and were used as received unless otherwise stated. Aqueous solutions were constituted in >18 MΩ/cm H2O. Tissue uptake of radioactivity is specified in standardized uptake values (SUV), defined as the product of the percentage of injected dose per gram of tissue (% ID/g*100) and the body weight (g) of the subject. Unless otherwise stated, all values are specified as mean±standard deviation.
Targets were prepared by electrolytic deposition of isotopically enriched Fe-54 metal (<100 mg) on Ag disc substrates (0.5 mm thick, 19 mm diameter). Briefly, Fe-54-enriched metal (99.93%, Isoflex USA, San Francisco, Calif.) was dissolved in 2-5 mL of 6 M HCl. To this solution, 100 μL of 30% H2O2 was added to promote the Fe(III) oxidation state. This solution was taken to near dryness (<1 mL), before adding 15 mL of saturated ammonium oxalate solution (stock solution stored with ˜g Chelex® 100 resin to minimize trace metal impurities). Approximately 100 mg of L-ascorbic acid was added to this solution to promote the reduction of Fe(III) cations during electrodeposition. This solution was adjusted to pH˜3.0 using 6 M NaOH or 6 M HCl and transferred to a cylindrical plating cell. A platinum wire anode was positioned approximately 1 cm above the silver disc substrate, and a potential of 7.0±0.1 V was applied corresponding to an initial current of 0.09±0.01 A (115±13 mA/cm2). Electrical current and pH were measured at multiple time-points during electrodeposition. 20 μL aliquots of the plating solution were also collected at multiple time-points for Fe-concentration measurements by microwave plasma atomic emission spectroscopy (MP-AES, Agilent Technologies, Santa Clara, Calif.). When electrodeposition had completed as determined by the electrolyte becoming colorless (˜24 hours), targets were dried and weighed to determine the plated Fe-54 mass.
Targets were irradiated by 16 MeV protons (PETtrace 800, GE Healthcare, Chicago, Ill.) with water-jet cooling on the rear target face. Beam currents of up to 60 μA were applied without changes in target appearance. Following irradiation, the short-lived Co-54 (t1/2: 1.5 min) impurity was allowed to decay for 10 minutes before dismounting the target. Activities were quantified by efficiency-calibrated high-purity germanium (HPGe) gamma spectroscopy, and end of bombardment (EoB) decay correction was performed using the nominal Mn-51 half-life (45.6 min).
Following irradiation, targets were placed in a cylindrical dissolution cell, whereby an o-ring sealed against the front of the target face around the electrodeposited and irradiated Fe-54 material. After the addition of 2 mL of 11M HCl, the reaction vessel was brought to 80° C. Dissolution was found to be complete in less than 20 minutes. To this solution, 1.8 mL H2O+0.2 mL 30% H2O2 was added before transferring to a 15 mL (1.5 cm diameter) AG-1×8 strongly-basic anion exchange column which had been equilibrated with ˜5 column volumes of 5 M HCl. Using 5 M HCl as mobile phase, the first 5 mL of eluent were discarded. The following 10 mL, containing the Mn-51 product, were collected in a pear-shaped rotary evaporator flask. The Mn-51 product was taken to dryness under reduced atmosphere, and the resulting 51MnCl2 residue was redissolved in ˜500 μl of pH 6.5 0.01 M NaOAc buffer. The enriched Fe-54 target material was recovered from the separation column in 30-50 mL of 0.1 M HCl, which was subsequently taken to dryness (ferric chloride) by boiling under N2 gas flow.
The Mn(II) oxidation state following separation was confirmed by thin-layer chromatographic techniques. Residual iron impurities in the final Mn-51 product were quantified by MP-AES analysis. An effective specific activity was measured by competitive DOTA chelation (room temperature, 0.15 M NaOAc, pH˜6.0, 1 hour) followed by silica thin-layer chromatography (0.25 M NH4OH). The mass of DOTA required to bind 50% of a sample's activity was interpolated from the resulting sigmoidal binding curve, and effective specific activity was calculated as the amount of activity divided by twice this mass.
All animal studies were conducted under a protocol approved by the University of Wisconsin Institutional Animal Care and Use Committee. Non-fasted healthy ICR mice (Envigo, Indianapolis, Ind.) were divided into two groups. Mice in the first group (n=2) were anaesthetized by isoflurane (4% induction, 1% maintenance), tail-vein catheters were affixed, and mice were placed on the microPET/CT bed in a prone position (Inveon, Siemens Preclinical Solutions, Knoxville, Tenn.). Dynamic PET acquisition was started and 51Mn2+ was administered in a rapid bolus (3.3 MBq, 200 μl, 10% 0.01 M NaOAc/90% PBS) through the tail-vein catheter. Sixty minutes of dynamic PET data were acquired following 51Mn2+ administration. Due to the impact of volatile anesthetics on voltage-dependent calcium channel (VDCC) activation, the second group (n=3) received an intravenous (I.V.) bolus of 51Mn2+ (1.6 MBq, 200 μL, 10% 0.01 M NaOAc/90% PBS) while awake. 60 minutes post-injection mice were anaesthetized by isoflurane and a 10 minute static PET scan acquired. Following imaging, mice were immediately sacrificed by CO2 asphyxiation, and organs were extracted. Ex vivo biodistribution measurements were performed by gamma counting (Wizard 2480, PerkinElmer, Waltham, Mass.).
Dynamic PET data were binned into 46 frames (12×5 s, 6×10 s, 6×30 s, 6×150 s, 6×300 s) and frames were reconstructed using non-scatter-corrected 3D ordered-subset expectation maximization followed by maximum a posteriori reconstruction (OSEM3D/MAP). Static PET data were reconstructed into a single frame by OSEM3D/MAP.
Due to the rapid blood clearance of Mn2+, OLINDA (Organ Level INternal Dose Assessment) dosimetry calculations were performed assuming instant compartment localization with organ activity fractions equal to those measured by ex vivo biodistribution herein. Based on the previously measured lengthy organ residence times of Mn2+, it was also assumed that the effective organ clearance half-life (Ter) was equal to the radioactive half-life of Mn-51 (t1/2: 45.6 min). It was also assumed that Mn-51 injections were 100% radionuclidically pure. In regards to the daughter isotope, Cr-51 (t1/2: 27.7 d), it was assumed that the activity remained in same organ compartments as the parent Mn-51 biodistribution without biological clearance. Standard radiation weighting factors were used (γ=1, β=1). Source-organ integrated decays for Mn-51 and Cr-51 are tabulated in Table 2. Based on these assumptions, effective dose (ED) and effective dose equivalent (EDE) (units of mSv/MBq) were calculated for a standard adult male and female.
Electrodeposition was found to be complete in approximately 24 hours with residual iron concentration dropping to <0.04 mg/mL (˜0.5 mg Fe-54 unplated). Changes in plating metrics during electrodeposition are shown in
Total chemistry duration including dissolution, separation, dry-down, and final formulation was found to be approximately 90 minutes. Decay-corrected Mn-51 yield was 67.3±12.6% (n=3). Recovery yields could be improved by collecting more than 10 mL of eluent at the expense of increased separation and dry-down duration. For targets of thicknesses 46-64 mg/cm2 (n=3) irradiated by 30 μA of 16 MeV protons for one hour, end of chemistry (EoC) yield was found to be ˜185-370 MBq (n=3).
Final iron impurity masses for three production trials are listed in Table 3, along with corresponding separation factors.
Fe-54 recovery efficiency between productions was found to be 94.3±4.2% (n=3). The final Mn-51 product, decay-corrected to EoB, was found to be >99.9% radionuclidically pure by HPGe gamma spectroscopy with the Cr-51 daughter being the largest impurity (0.08%/). Trace radionuclidic impurities are listed in Table 4. An EoB effective specific activity of 7.4 GBq/μmol (1.9 GBq/μmol at EoC, n=1) was measured by titration with DOTA.
51Mn
52Mn
51Cr
55Co
56Co
57Co
Rapid Mn-51 accumulation in the heart, liver, kidneys, pancreas, and salivary glands was observed in ICR mice (n=5) following a rapid intravenous bolus injection. PET time-activity curves (TACs) are shown in
Delineation of the pancreas from the surrounding organs (e.g., the kidneys) was readily achieved in static PET images (
Good agreement was observed between in vivo PET quantification and ex vivo gamma counting in all tissues with the exception of the heart. Because tissues are rinsed and wicked dry prior to weighing and gamma counting, this discrepancy in measured heart uptake is likely due to the inclusion of low-activity blood mass in heart PET ROIs. Intersubject biodistribution variability was found to be minimal when using the SUV uptake metric despite highly varied subject weights (37.6, 48.3, and 22.1 g). As expected, greater intersubject biodistribution variability was observed when using the % ID/g uptake metric.
Mn-51 was found to have an EDE of 0.0362 mSv/MBq and 0.0422 mSv/MBq for the standard male and female human model respectively. The daughter isotope Cr-51 was found to have an EDE of 0.267 mSv/MBq and 0.324 mSv/MBq for the standard male and female model respectively. OLINDA dosimetry predictions for a typical clinical dose (370 MBq, 10 mCi) are listed in Table 5.
51Mn
51Cr
Manganese is an essential trace element in mammalian biology and has many prospective applications as an imaging agent in medicine. Mn-51 is well suited for clinical PET based on its decay characteristics.
The electrodeposition method described in this example study was effective for the quantitative reduction of 54Fe(III) to Fe-54 metal, with the electroplated iron metal being strongly adhered to the silver disc substrate. From
The fabricated Fe-54 targets were robust, withstanding relatively high beam currents (16 MeV, 60 μA) without changes in appearance. The target thicknesses (˜45-65 mg/cm2) and irradiation parameters (30 μA for 1 h) used in this example study were sufficient to provide enough EoC activity (˜185-370 MBq) for several small animal studies or approximately one human study. EoC yield could readily be increased to 1.5-2.0 GBq by employing target thicknesses of approximately 100 mg/cm2 and irradiating with a beam current of 60 μA for two hours. Based on these yields, a chemistry duration of ˜90 minutes is sufficiently short for production purposes. However at institutions without solid-target capabilities, a solution-target of 54Fe(NO3)2 or 50Cr(NO3)3 could provide elegant alternative production routes. Although the chemical isolation of Mn-51 from bulk iron metal is simpler than Mn-51 from bulk chromium, the production cross section for 50Cr(d,n) is significantly higher than 54Fe(p,α) which may help compensate for the reduced target atomic fraction in solution targets.
PET imaging of pancreatic beta cells with 51MnCl2 appears promising due to the rapid blood clearance and significant pancreatic accumulation. Further studies are needed to determine the feasibility and optimal study methodology for functional beta cell mass quantification by 51Mn-PET. To this end, it is contemplated that non-specific exocrine uptake can be quantified by co-injection of VDCC blocking agents, such as nifedipine. Other positron-emitting divalent metals such as 63Zn2+ (t1/2: 38.5 min β+: 92.7%, Eβave: 0.92 MeV) may also prove useful for beta cell related investigations, as VDCCs are permeable to Zn2+ and significant 63Zn2+ pancreatic uptake has been observed in mice in other studies.
The heart blood-pool clearance half-life of 51Mn2+ found in this example study (7.7±0.7 s) was rapid, suggesting first-pass tissue localization kinetics. Rapid blood clearance and stable accumulation offers experimental flexibility with regards to PET imaging duration and timing following tracer administration. Tracer kinetics such as these also support the use of the SUV uptake metric for 51Mn-PET studies, as tracers without significant tissue clearance (e.g., [18F]-FDG) lend themselves well to such analytic methods. Furthermore, the rapid blood clearance of 51Mn2+ may enable multiple-injection protocols within a single patient study. Techniques such as these may prove useful in beta cell mass (BCM) quantification studies for the subtraction of non-specific exocrine pancreas uptake by stimulation or blocking (e.g., through glibendamide or nifedipine) of beta cell VDCCs following baseline imaging. On the other hand, the pulsatile nature of calcium transport may increase test-retest variability for bolus injection techniques. This effect could possibly be mitigated by administering 51MnCl2 as an intravenous infusion over 5-15 minutes.
The mean positron energy emitted during the decay of Mn-51 (962 keV) is significantly higher than that of F-18 (250 keV) or 52gMn (242 keV), which may lead to poorer spatial resolution in PET images. Regardless, the resolution of Mn-51 has still proven to be sufficient for whole-organ-ROI microPET studies, and positron range is not typically the limiting factor of clinical PET resolution.
Mn-51 dosimetry appears favorable, even when accounting for the long-lived daughter Cr-51, and making the conservative assumption that this daughter is not biologically excreted. In this example study, a cumulative effective dose equivalent of ˜15 mSv for a 370 MBq Mn-51 PET study was calculated. This result was comparable to the average dose for an [18F]-FDG study of 14.1 mSv. This suggests that it would be possible to perform up to three repeat PET studies in healthy or type-I diabetic volunteers without exceeding the annual non-stochastic International Commission on Radiological Protection (ICRP) limit of 50 mSv for research subjects.
The present disclosure has described one or more preferred embodiments, and it should be appreciated that many equivalents, alternatives, variations, and modifications, aside from those expressly stated, are possible and within the scope of the invention.
This application claims the benefit of U.S. Provisional Patent Application Ser. No. 62/475,571, filed on Mar. 23, 2017, and entitled “METHODS FOR QUANTIFYING PANCREATIC BETA CELL FUNCTION AND MASS PROPERTIES WITH RADIOMANGANESE POSITRON EMISSION TOMOGRAPHY,” which is herein incorporated by reference in its entirety.
This invention was made with government support under CA169365 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62475571 | Mar 2017 | US |
