Positron Emission Tomography Using 64-Copper as a Tracer for the Study of Copper-Related Disorders

Copper is a vital catalytic cofactor that drives multiple important biochemical processes essential for life, and defects in copper homeostasis are directly responsible for human diseases. Mutations in the ATP7A or ATP7B genes, encoding copper-transporting ATPases, cause Menke's and Wilson diseases, respectively. Menke's disease (ATP7A) is a rare lethal disorder of intestinal copper hyperaccumulation with severe copper deficiency in peripheral tissues and deficits in copper-dependent enzymes¹. Wilson disease (WD) (ATP7B) is a rare disease characterized by the inability to excrete excess copper to bile, resulting in copper overload and subsequent organ damage, most severely affecting the liver and brain².

Studies on copper metabolism have utilized radiolabeled copper (usually 64-copper [⁶⁴Cu] or 67-copper) for decades, and these studies have proven invaluable for our understanding of mammalian copper metabolism, including absorption site and excretion pathways^3,4,5,6. Previously, gamma counters were used to detect the radioactive signal with a limited anatomical resolution, but recently, ⁶⁴Cu positron emission tomography (PET) combined with computed tomography (CT) or magnetic resonance imaging (MRI) has been introduced in both human and animal studies. Today, PET scanners have such a high sensitivity that it is possible to track ⁶⁴Cu for up to 70 h after injection. The long half-life of 12.7 h for ⁶⁴Cu allows for the long-term assessment of copper fluxes. This improvement in resolution has just recently entered the field of copper studies, and studies on normal and pathological copper metabolism, as well as studies evaluating the impact of specific treatments, are starting to emerge. Additionally, the introduction of whole-body PET scanners with an extended field-of-view will further enhance the sensitivity of these examinations.

This methodological paper aims to enable clinicians and scientists to add ⁶⁴Cu PET CT/MRI to the existing repertoire of tools as a robust and easy-to-use method for assessing copper metabolism in a manner comparable between nuclear medicine departments. The production of ⁶⁴Cu copper can be carried out using different methods and is usually performed at special facilities. Among the nuclear reactions, the ⁶⁴Ni (p, n) ⁶⁴Cu method is widely used, since a high production yield of ⁶⁴Cu can be obtained with low energy protons in this route^7,8. A detailed description of the production methods is out of the scope of this work, and availability will differ by country and region.

In this article, we first describe the preparation of the necessary radiochemistry and the tracer. Then, the principles for preparing the PET/CT or PET/MRI scanners are demonstrated.

A few clinical trials using this ⁶⁴Cu PET/CT or PET/MRI protocol have been approved by the Regional Ethics Committee of Region Midt, Denmark [1-10-72-196-16 (EudraCT 2016-001975-59), 1-10-72-41-19 (EudraCT 2019-000905-57), 1-10-72-343-20 (EudraCT 2020-005832-31), 1-10-72-25-21 (EudraCT 2021-000102-25), and 1-10-72-15-22 (EudraCT 2021-005464-21)]. Written informed consent was obtained from the participants at enrollment. The inclusion criteria for all participants were age >18, and for females the use of safe contraception. The exclusion criteria for Wilson disease patients were decompensated cirrhosis, a Model for End-stage Liver Disease (MELD) score >11, or a modified Nazer score >6. The exclusion criteria for all participants were a known hypersensitivity to ⁶⁴Cu or other ingredients in the tracer formula, pregnancy, breastfeeding, or a desire to become pregnant before the end of the trial.

1. Preparation of ⁶⁴CuCl₂

1. Dissolve solid ⁶⁴CuCl₂ in hydrochloric acid (0.1 M) and add sodium acetate buffer (0.5 M) to increase the pH to ~5. Formulate with saline and filter sterilize the solution by passing it through a 0.22 µm filter (see Table of Materials).
   NOTE: Sodium acetate buffer (0.5 M) is produced from sodium acetate trihydrate and sterile water that is passed through a 0.22 µm sterilizing filter.
2. For quality control of the produced ⁶⁴CuCl₂ solution, perform pH measurement, bacterial endotoxin testing, radiochemical purity determination, and radio nuclidic identification^7,8.
3. Store the product in a lead container at room temperature and keep it in quarantine until all quality control specifications have been satisfactorily met.
   NOTE: For the present study, ⁶⁴CuCl₂ was produced with a radio nuclidic purity ≥99% and a radiochemical purity ≥95%. Solid ⁶⁴CuCl₂, used as starting material, was obtained from a commercial source (see Table of Materials).

2. Preparation of PET scanner

1. Perform a quality check (QC)⁹ on the scanner, following the manufacturer's protocol (see Table of Materials).
   NOTE: QCs must be performed daily in the morning before patient scans.

3. Drawing of tracer for intravenous (IV) injection and per oral (PO) administration

1. Wear plastic gloves and remove the lid from the lead container.
2. Use long tweezers to disinfect the rubber membrane of the tracer-containing glass bottle inside the lead container with a disinfection swab.
3. Use tweezers to insert a short cannula (~0.5 mm x 16 mm) into the membrane to avoid spill-over from the vacuum inside the bottle.
4. Use tweezers to insert a longer cannula to draw from. This cannula should be long enough to reach the bottom of the bottle (usually 50 mm).
5. Ensure that the dose calibrator (see Table of Materials) is calibrated for ⁶⁴Cu. Calculate an approximate volume to draw for the first draw.
   NOTE: From the chemical quality control reports, the activity amount and volume of the liquid will be available, allowing for the calculation of an approximate volume to draw.
6. Wear plastic gloves, insert an appropriately sized plastic syringe into the long cannula, and draw the calculated volume. This volume will depend on the concentration of ⁶⁴Cu in the product and how much ⁶⁴Cu is decided for the protocol (see Dose calculations under the representative results).
7. Use tweezers to hold the cannula while moving the syringe to the dose calibrator to measure the radioactivity.
8. Keep drawing until the appropriate radioactivity amount is reached. Approximately 5% of the tracer will remain in the syringe and cannula after injection.
   NOTE: The ⁶⁴Cu should not be diluted in salt water, as the tracer may precipitate. Thus, the syringe cannot be rinsed with saline water after the injection (this is not relevant for PO administration).
9. With the tweezers, apply a cannula with a cap (~16 mm cannula) to close the syringe and store it in a lead container until application.

4. Application of the tracer

1. IV injection
   1. Insert an intravenous cannula (~22 G, 25 mm), preferably in a cubital vein, and rinse with saline water to ensure correct placement.
      NOTE: A worksheet with the participant's name, a stamp or signature for tracer quality control release, and time points and radioactivity for drawing, injection, and leftover tracer should be available.
   2. Measure the radioactivity in the syringe using the dose calibrator available and note the time and activity on the worksheet.
   3. Transport the syringe in a lead container to the participant's bedside.
   4. If any spill-over occurs from the injection, place a napkin under the participant's elbow so the spilled radioactivity can be measured.
   5. With tweezers, remove the cap/cannula from the syringe, and with plastic gloves, connect the syringe to the IV access. Note the time on the worksheet and inject in one steady movement.
      NOTE: As mentioned earlier, the syringe should not be rinsed with saline as the tracer may precipitate.
   6. Remove the syringe from the IV access, put on the cap/cannula, and place it in the lead container with the napkin if necessary.
   7. Rinse the IV access through with saline water.
   8. Note the time and leftover radioactivity in the syringe on the worksheet.
      NOTE: The injected activity is calculated as the difference between the syringe activity before and after injection, but using the PET scan protocol to correct for decay. Thus, all three time points (draw, injection, and leftover measurements) and the measured radioactivity at the draw and leftover measurements are entered into the PET scan protocol when the participant is scanned (see step 5).
   9. Dispose of the leftover material appropriately, according to institutional safety regulations.
   10. Remove the IV access. In case any allergic reactions appear, leave the IV access in for 30 min.
2. Oral administration
   NOTE: A worksheet with the participant's name, a stamp or signature for tracer quality control release, and time points and radioactivity for drawing, administration, and leftover tracer should be available.
   1. In a disposable and soft plastic cup, pour around 100 mL of water or cordial; the ⁶⁴Cu is tasteless. A disposable plastic straw and a small disposable plastic bag should be available.
   2. Measure the radioactivity in the syringe using the dose calibrator available and note the time and activity on the worksheet.
   3. Transport the syringe in a lead container to the participant's bedside. The participant should be seated in a bed or chair.
   4. Remove the cap/cannula from the syringe with tweezers and, wearing plastic gloves, inject the tracer into the cup, being careful not to spill any. Draw up a bit of the water/cordial and inject it into the cup again.
   5. Place a plastic straw in the cup (this is to minimize the risk of spill-over when the participant drinks).
   6. Note the time on the worksheet and let the participant drink. The cup should be as empty as possible.
   7. Put the empty cup and straw in the disposable plastic bag with the empty syringe and place them in the lead container.
   8. Note the time and measure the leftover radioactivity in the syringe. Note in the worksheet.
      NOTE: The injected activity is calculated as the difference between the syringe activity before and after injection, but using the PET scan protocol to correct for decay. Thus, all three time points (draw, injection, and leftover measurements) and the measured radioactivity at the draw and leftover measurement are entered into the PET scan protocol when the participant is scanned (see Scan).
   9. Dispose of the leftover material appropriately, according to institutional safety regulations.
      NOTE: Observing the participant for acute allergic reactions for 30 min after the intake may be appropriate.

5. PET scans

1. Place the participant in a supine position in the scanner.
2. Perform overview CT or MR scanning to plan the specific region to be examined during the PET scan.
3. Note the time of the draw, injection, and leftover measurement, and the radioactivity at the draw and leftover measurement in the PET protocol.
4. Perform PET scanning following the steps below.
   NOTE: The PET scan protocol must be standardized with regard to scan duration and image reconstruction parameters for all participants in the same study; published reports should be followed^10,11,12.
   1. Perform static PET scans with a scan time of 4.5 min/bed position for up to 24 h after tracer administration, and 10 min/bed position for up to 68 h after tracer administration (for further elaboration, see Scan under the representative results).
      NOTE: During dynamic PET scanning, decay is continuously recorded and subsequently segmented into a frame structure. This allows for selection of frames from short time intervals to emphasize the dynamics of ⁶⁴Cu distribution, and frames from longer time intervals to prioritize sensitivity. Typically, shorter intervals are selected right after injection and gradually increased thereafter¹⁰.

6. Image reconstruction

1. Reconstruct the images using the best available corrections for attenuation, scatter, time-of-flight, and point-spread function.
   NOTE: The image reconstruction parameters must be carefully selected to optimize the image properties, such as signal recovery and signal-to-noise. For multicenter studies, it is critical to standardize the image quality between centers.

7. Data analysis

NOTE: The present study describes a simple method to quantify ⁶⁴Cu content in the liver. The PET signal is measured as standard uptake value (SUV), the tissue radioactivity concentration adjusted for participant weight injected activity and/or kilobecquerel (kBq) per mL of tissue.

1. Download data to a suitable program, for example, Dicom files, to PMOD.
   NOTE: There are likely many different programs to analyze PET images, such as Hermes or PMOD (see Table of Materials).
2. Adjust the CT/MR scan tones to differentiate the anatomical structures.
3. Ensure that the anatomical scan and PET scan are overlapping.
4. Working in the horizontal plane with the best MRI or CT scan, localize the liver and the big structures.
5. Place an appropriate volume of interest (VOI) or multiple VOIs in the liver.
   NOTE: A VOI is a defined area of tissue where the SUV is measured. A VOI consists of multiple regions of interest (ROIs), which are tissue areas in one plane. Many programs have spherical VOIs as a pre-setting, meaning multiple ROIs (one in each plane) do not have to be drawn to constitute a VOI. The right liver lobe tends to be more homogenous, and thus a good position to place VOIs.
6. Place multiple VOIs in the right liver lobe in different horizontal planes to achieve the most precise measure of activity, as the SUV may vary somewhat (~5%) in the right liver lobe. Calculate the mean SUV of these VOIs.
7. To quantify the SUV, for example, in the entire liver, draw ROIs covering the whole liver volume in each plane for dosimetry studies.
   NOTE: Avoid big structures such as arteries and veins when using this method.

Dose calculation
Based on dosimetry calculations, the effective radioactivity dose for IV administration is 62 ± 5 µSv/MBq tracer¹⁰. Thus, a 50 MBq dose is recommended depending on the time frame. Up to 75-80 MBq is applicable for longer examinations and provides good-quality images without exceeding an ethically approved dose. The effective dose for oral administration is 113 ± 1 µSv/MBq tracer, due to intestinal accumulation of the tracer. Thus, a lower dose needs to be considered, and for up to 24 h post-injection, 30 MBq is sufficient to yield high-quality images. Fertile female participants should always be asked for a negative pregnancy test before tracer application.

Scan
For very long examinations, performed to follow the ⁶⁴Cu biodistribution and kinetics for hours or days, the PET examination is performed as multiple separate static PET scans. This allows the patient to rest between the PET examinations. The duration of each PET examination is adjusted to achieve the best image quality (i.e., the scan time is prolonged as the injected tracer decays). An example of scan times providing good-quality images is 4.5 min/bed position for up to 20 h after tracer administration and 10 min/bed position for up to 68 h after tracer administration. Longer scan times may provide even better image quality, but too-long scans are unfeasible and uncomfortable for the patient. Thus, the length of the scans is limited by practicalities.

Data analysis
SUV is an excellent measure to compare individuals (because of the weight adjustment) and to compare the same individuals before and after an intervention. A standard deviation of the SUV in the VOI is available from the data analysis program (e.g., PMOD). This standard deviation increases with time after injection because the noise increases.

Figure 1 shows ⁶⁴Cu in the body 6 h and 20 h after IV injection of ~70 MBq tracers in a healthy subject and a subject with WD¹⁰. The images are qualitatively easy to interpret as the ⁶⁴Cu is quickly visible in the gallbladder (difficult to see in the figure), small intestine, and later in the colon, while it accumulates in the liver in the patient. The gut is also visible on the patient's scan, however this is not from ⁶⁴Cu in the gut lumen but rather from intestinal blood vessels. The gut is seen by the ⁶⁴Cu being more homogenously distributed along the entire gut segment, whereas in healthy subjects, the ⁶⁴Cu is visible in segments with higher signals. The ⁶⁴Cu content in the liver was further quantified by placing five spherical VOIs with a diameter of 10 mm in different planes in the right liver lobe, yielding a mean SUV in the organ for each participant, then calculating the group's mean SUV for comparison between groups.

Figure 1: PET scan showing ⁶⁴Cu distribution in healthy and WD subjects after IV administration. This figure shows ⁶⁴Cu in the body 6 h and 20 h after IV injection of ~70 MBq tracers. Please click here to view a larger version of this figure.

Figure 2 shows the results of ⁶⁴Cu scans with the orally administered tracer in two individuals. Both are WD patients, but the bottom individual is under zinc treatment, demonstrating that zinc treatment reduces copper uptake in the intestines and thus to the liver; this is a well-known effect of zinc treatment¹³. While orally administered tracer is the physiological way of ingesting copper, it may be difficult to use for diagnostics, as only 50% of the ⁶⁴Cu is taken up from the intestines to systemic circulation (most of the tracer goes to the liver). However, to demonstrate the effects of pharmacologic drugs on copper uptake, which may be of great interest in WD, the method has shown to be valuable¹¹. This can be seen in Figure 3, in which the same individual has been scanned using oral ⁶⁴Cu before and after 4 weeks of treatment with zinc¹¹. The study hypothesis was to quantify zinc's effect on blocking intestinal copper uptake by estimating the copper content in the liver. The study was performed with different zinc salts and dose regimens and demonstrates the method's qualities in testing treatment effects. The method's ability to quantify other treatment effects in animals and humans is being tested.

Figure 2: PET scan showing ⁶⁴Cu distribution in two WD patients after per oral administration. The patient in the upper panel is without zinc treatment, and the patient in the lower panel is on zinc treatment. Note the signal difference in the liver. Graph depicting liver SUV. Please click here to view a larger version of this figure.

Figure 3: The effects of pharmacologic drugs on copper uptake. PET/CT scan using orally administered ⁶⁴Cu before (A) and after (B) 4 weeks of zinc treatment. The participant is a healthy individual (notice the ⁶⁴Cu in the gallbladder, which would not be seen in a WD patient). Zinc treatment reduced ⁶⁴Cu content in the liver to around 50% of the pre-treatment content in the group (10 participants). Please click here to view a larger version of this figure.

The method is like any other PET method, but the long half-life of 12.7 h offers the opportunity to investigate long-term copper fluxes (we have good results from up to 68 h after IV tracer injection). All steps in the protocol must be handled by personnel familiar with PET, although they are no more critical than any other PET examination.

Troubleshooting
Because we often use ⁶⁴Cu for long-term investigations, the PET signal will be noisier than usual. This is important to remember when quantifying PET signals, particularly in smaller organs such as the gallbladder. The signal in the gallbladder will be difficult to distinguish from spill-over from the liver and colon. In this case, smaller VOIs centrally in the organ are the most reliable.

The amount of ⁶⁴Cu in the liver, from our experience, tends to vary between individuals despite IV injection (a fairly large variance in tracer uptake from the gut must be expected with an orally administered tracer). This limits the comparisons between individuals and calls for the use of ratios instead of definite numbers. If per oral tracer administration is preferred, keeping trial subjects on a standardized diet for a minimum of 24 h before the tracer intake is recommended to limit intra-individual differences, as different foodstuffs may interfere with copper and thus with ⁶⁴Cu uptake¹¹.

Limitations
When the ⁶⁴Cu PET method is used, it is assumed that the "hot" copper (⁶⁴Cu) acts like the "cold" copper in the body. However, this is not certain, and we thus cannot determine if the "hot" copper is treated differently in the body. From the current results, however, we believe that "hot" copper acts like "cold" copper. An increase in blood radioactivity after 20 h is observed in healthy individuals, indicating that the ⁶⁴Cu is built into ceruloplasmin. This increase is not seen in WD patients, who cannot build copper into the copper-carrying protein because of their disorder. This and the lack of tracer excretion in patients point toward ⁶⁴Cu acting as "cold" copper.

Although 68 h is a long time to follow a radioactive tracer, it should still be considered a temporary image of what happens to copper in the body. An example is that even though stalled ⁶⁴Cu excretion is seen in individuals who are heterozygotous for the WD gene, and thus more ⁶⁴Cu in the liver after 20 h, they do not have liver disease because, in the long term, they do not accumulate copper.

So far, it is not known if there is a correlation between short-term copper accumulation (up to 68 h) and long-term copper accumulation in the liver and other organs. Thus, the method cannot be used to determine disease severity or the long-term effects of pharmacological agents. However, the method is very useful in determining the short-term effects of treatment. It may be used to test whether treatment increases biliary or urinary excretion up to 68 h after copper intake, or if a treatment decreases intestinal copper uptake.

Significance
Experiments with ⁶⁴Cu in WD is not a new technique. In fact, IV administration of the tracer and blood measurements of radioactivity goes back to the 1950s¹⁴. Today, high-resolution PET scanners and combination with CT or MR provide a unique opportunity to investigate ⁶⁴Cu distribution in the whole body. With dynamic PET, the kinetic properties of the tracer can further be elucidated. So far, due to the limited field-of-view of PET scanners, conducting kinetic analyses of copper's biodistribution throughout the body has not been feasible. Currently, dynamic uptake has been restricted to the liver and upper abdomen, but the advent of whole-body scanners will enable the simultaneous investigation of larger areas. This will facilitate examination of the initial period after the injection of ⁶⁴Cu in multiple organs, but given that late time points after injection are more relevant for copper-related disorders, whole-body scanners are expected to be more significant due to their heightened sensitivity. This allows for high-quality imaging even at low radioactivity levels, surpassing current scanners' capabilities.

Future applications
In humans, the technique has shown potential in diagnosing WD¹⁰ and quantifying the effect of different treatments on copper uptake¹¹. In animals, the method has proven to be able to show the effect of gene therapy of WD by quantifying the hepatic retention of ⁶⁴Cu as well as fecal excretion and changes in blood kinetics¹⁵. In the future, it is expected that ⁶⁴Cu PET/CT or PET/MR will be seen in a clinical setting for both diagnosis and treatment evaluation in WD. The method is also highly likely to be part of many clinical trials involving new therapies for WD, especially gene therapy, in which fecal excretion of the IV injected tracer could be a surrogate marker of effect¹⁵. There is currently no good data for brain ⁶⁴Cu uptake available, but this would be highly relevant for clinical studies in WD.

The technique has not been explored in Menke's disease yet, but could potentially show copper uptake from the gut and copper uptake into the brain as a treatment effect. The technique may also have potential in neurodegenerative diseases such as Alzheimer's disease, where copper metabolism may be altered¹⁶.

It is worth noting that ⁶⁴Cu is becoming widely available in the US with the increasing use of ⁶⁴Cu-Dotatate in neuroendocrine tumor (NET) diagnostics. Furthermore, ⁶⁷Cu is showing potential in cancer theranostics; thus, this tracer may also become more available.