Summary

Bisphosphonates (BPs) are compounds with high affinity for solid calcium minerals such as hydroxyapatite (HAp), the primary inorganic component of bone tissue. BPs accumulate particularly in areas of high mineral turnover, such as bone metastases, and have been the mainstay of medical imaging of bone diseases since the 1970s, most commonly in the form of [^99mTc]Tc-MDP using gamma-scintigraphy/SPECT imaging.
Technological advances in positron emission tomography (PET) have ushered in renewed interest in the development of BP-based imaging agents due to its high sensitivity and spatial resolution. In recent years, a flurry of new BP tracers using the generator-produced positron emitter ⁶⁸Ga (t_1/2 = 68 min), have been reported, mainly focusing on applications for the detection of bone metastases. However, the current most-used tracer for the PET imaging of bone metastases in the clinic is [¹⁸F]NaF, which functions due to the fluoride ion’s ability to displace the hydroxyl group in the structure of HAp.
Previously, the authors have demonstrated that the binding mode of [¹⁸F]NaF leads to binding specificity towards HAp over other calcium minerals. However, BPs—which bind through the interaction of the phosphonate groups with calcium ions—have a much broader range of calcium mineral affinity. The prevalence of HAp in bones makes [¹⁸F]NaF an excellent tracer for the imaging of bones. On the other hand, reports on the mineral composition of extraosseous calcification (EC), which is defined as “deposition of calcium in tissues outside of bone” and includes conditions such as vascular calcification, are diverse and contradictory. These calcifications are often euphemistically called HAp as a blanket term for all forms of apatite or solid calcium-containing mineral. However, the general consensus in the literature is that early calcification begins as microcalcifications of amorphous calcium phosphate and whitlockite and, as the calcification progresses, HAp crystals develop and merge into larger sheets or plaques of macrocalcification. However, this is dependent on a number of factors including the location of the calcification and its underlying cause. This consensus is oversimplified and a range of calcium salts may be observed including HAp, apatite, (amorphous) calcium phosphate, whitlockite, calcium oxalate and calcium carbonate. Various studies have demonstrated the presence of calcification in a wide variety of diseases, including atherosclerosis, age-related macular degeneration, Alzheimer’s disease, muscular dystrophy, various cancers, kidney stones and chronic kidney disease (CKD).
Nonetheless, [¹⁸F]NaF is the only clinically used PET tracer for the imaging of extraosseous calcification, although a recent study has demonstrated the feasibility of the PET BP [⁶⁸Ga]Ga-NODAGA^ZOL for the imaging of EC in atherosclerotic plaques. Given the potentially diverse forms of calcium mineral present in such calcification, the authors hypothesised that a HAp-selective tracer such as [¹⁸F]NaF is not the most appropriate imaging agent for a condition in which HAp may not be the most common form of calcium mineral. To test this hypothesis, the authors compared the performance of [¹⁸F]NaF with the BP-based conjugate of the ⁶⁸Ga chelator tris(hydroxypyridinone) (THP)—[⁶⁸Ga]Ga-THP-Pam, using a rat model that develops macro- and microcalcification across several major organs, and highlighted the differences in imaging results that stem from the mineral composition of the calcification.

Results from the nanoScan PET/CT

On day 11, rats were anaesthetised by inhalation of isoflurane (1.5–4% in oxygen) and the tail vein was cannulated using sterile saline. Each rat was injected intravenously with [⁶⁸Ga]Ga-THP-Pam (100 ± 20 μL, 1.0–6.6 MBq). The rat was maintained under anaesthetic on a warm bed to maintain body temperature for 30 min. The rat was placed in a Mediso nanoScan® PET/CT scanner, where anaesthesia was maintained, and the bed was heated to maintain normal body temperature and CT (55 kVp) was performed. At 1 h post-injection, 1 h PET acquisition (3 × 20 min fields of view, 1:5 coincidence mode; 5-ns coincidence time window) was performed. On day 12, the rats were anaesthetised by inhalation of isoflurane (1.5–4% in oxygen) and the tail vein was cannulated. Each rat was injected intravenously with [¹⁸F]NaF in sterile saline (100 ± 15 μL, 2.7–11.7 MBq) and imaged at 1 h post-injection on the PET/CT scanner using the same procedure used on day 11. At the end of the scan, the animal was culled at 2 h post-injection for ex vivo biodistribution studies. Additionally, non-imaging rats were anaesthetised by inhalation of isoflurane (1.5–4% in oxygen) and the tail vein was cannulated. The rats were injected intravenously with [⁶⁸Ga]Ga-THP-Pam (100 ± 5 μL, 3.9–10.9 MBq). The rats were maintained under anaesthetic on a warm bed to maintain body temperature for 2 h and culled at 2 h post-injection for ex vivo biodistribution studies. Organs were harvested, weighed, and counted with a gamma counter along with standards prepared from injected material. Organs and vasculature of interest (heart, lungs, stomach, kidneys, aorta, mesenterics and femoral artery) were fixed in 10% neutral-buffered formalin, after weighing, and subsequently embedded in paraffin for further analysis. For autoradiography, sections of abdominal aorta (approximately 1 cm long, centred on the branching points of the celiac and superior mesenteric arteries) from four rats (two fed the EC diet, two fed a healthy diet) were collected 2 h post-injection with the same volume (100 ± 10 μL) of the same stock of [⁶⁸Ga]Ga-THP-Pam during biodistribution studies. The aortas were placed under a PerkinElmer MultiSensitive Phosphor Screen (12.5 × 25.2 cm) for 5 min and the film was transferred to a Typhoon 8600 Variable Mode Imager. The results were processed using the open-source image processing and analysis package Fiji. The image was pseudo-coloured using the mpl-inferno scale.
PET/CT imaging with both radiotracers (Figs. 2, 3a,b, Table S1) show equal uptake in the skeleton (Fig. 3b) of both groups—extraosseous calcification diet (EC) and healthy diet—but differences in other organs. For example, both the stomach and kidneys of the rats fed the EC diet demonstrated a higher uptake of [⁶⁸Ga]Ga-THP-Pam (3.44 ± 0.69%IA—stomach; 2.21 ± 0.76—kidneys) than [¹⁸F]NaF (0.91 ± 0.24%IA—stomach; 0.19 ± 0.06%IA—kidneys). Uptake of both radiotracers in these organs in the healthy diet group were significantly lower. Significant increases in uptake were also seen between the two groups with both tracers in the heart, and with [⁶⁸Ga]Ga-THP-Pam in the lungs. Aortic and stomach calcification was visible by CT and detected with both radiotracers by PET (Fig. 3a,c).

• The authors have compared the PET imaging performance of a BP ([⁶⁸Ga]Ga-THP-Pam) to the clinical PET tracer ([¹⁸F]NaF) in a model of EC in rats. The results showed a significantly increased uptake of both tracers in several major organs in the EC group rats, with the increase in uptake being greater with [⁶⁸Ga]Ga-THP-Pam than with [¹⁸F]NaF.
• X-ray and histology studies confirmed the presence of calcification in these organs.
• Finally, the composition and morphology of the calcification were studied by SEM and EDX, which demonstrated that in organs where both radiotracers performed similarly by PET, the composition more closely matched theoretical HAp. Yet, in organs in which [⁶⁸Ga]Ga-THP-Pam detected calcification more sensitively than [¹⁸F]NaF, the composition varied away from theoretical HAp.

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