Biodistribution and radiation dosimetry of the novel hypoxia PET probe [¹⁸F]DiFA and comparison with [¹⁸F]FMISO

There is accumulating evidence that tumor hypoxia induces the expression of gene products that confer tumor propagation, malignant progression, and broad resistance to therapy [1]. Positron emission tomography (PET) is a useful clinical tool to visualize hypoxia in vivo. Among hypoxia PET tracers, [¹⁸F]fluoromisonidazole ([¹⁸F]FMISO) is the most extensively studied; however, its optimal acquisition time is 3–4 h after injection due to its slow specific accumulation in hypoxic tissue as well as its slow clearance from the plasma [2, 3]. Consequently, next-generation tracers such as [¹⁸F]fluoroazomycinarabinofuranoside ([¹⁸F]FAZA), [¹⁸F]fluoroerythronitromidazole ([¹⁸F]FETNIM), and [¹⁸F]-3-fluoro-2-(4-((2-nitro-1H-imidazol-1-yl)methyl)-1H-1,2,3-triazol-1-yl)propan-1-ol ([¹⁸F]HX4) have been developed and applied to clinical trials [4]. Like [¹⁸F]FMISO, these hypoxia probes are derived from the 2-nitroimidazole in their structures. However, they still have the clinical drawbacks of poor imaging contrast at acquisition time and limited reproducibility [5, 6].

We recently developed a new imaging tracer targeting tumor hypoxia, 1-(2,2-dihydroxymethyl-3-[¹⁸F]-fluoropropyl)-2-nitroimidazole ([¹⁸F]DiFA), to overcome the disadvantages of [¹⁸F]FMISO and obtain better contrast image quality in a shorter period of time. [¹⁸F]DiFA has lower lipophilicity and is thus expected to be excreted more rapidly via the urinary system. Moreover, as another advantage, we aimed to avoid enantiomers in the structure of [¹⁸F]DiFA to ensure efficient synthesis and quality control, which should make hypoxia imaging more readily available for clinical application. Shimizu et al. elucidated that the mechanism by which [¹⁸F]DiFA and [¹⁸F]FMISO targets hypoxia is the same [7].

In the present study, we first evaluated the radiation dosage, biodistribution, human safety, tolerability, and early elimination of ¹⁸F activity in urine after the injection of a single dose of [¹⁸F]DiFA in healthy volunteers. We then, for the first time, compared the hypoxia detectability of [¹⁸F]DiFA and [¹⁸F]FMISO in patients with malignant tumors.

In this work, we first evaluated the safety and dosimetric data of [¹⁸F]DiFA, a new [¹⁸F]FMISO-based derivative with stronger hydrophilicity, in healthy volunteers for its potential use as a hypoxia PET tracer. We found that [¹⁸F]DiFA caused no adverse effects after injection and had rapid clearance from the urine and reasonable biodistribution and dosimetry profiles in human subjects. In addition, in our comparison of the abilities of [¹⁸F]DiFA and [¹⁸F]FMISO to diagnose tumor hypoxia, we found that the diagnostic abilities were approximately equivalent, and good inter-observer reproducibility was observed even though there was a shorter time from the injection to the scan with the use of [¹⁸F]DiFA.

In our human study, high [¹⁸F]DiFA uptake/excretion was observed in the liver, muscle, and kidneys, whereas low uptake was observed in the lung and brain. These findings are similar to those for other hypoxia tracers, and the usefulness of evaluating neoplasms in the cranial should be investigated in detail. [¹⁸F]DiFA clearance from the blood pool is very rapid, and the accumulation in the blood pool was obscured at just 1 h after administration. In addition, since the distributions in the liver and intestines were relatively low, patients with hepatobiliary and colorectal cancers may be good candidates for hypoxia imaging using [¹⁸F]DiFA if the images are acquired at an appropriate time point.

[¹⁸F]DiFA had a higher rate of urinary excretion (86.4%) than other hypoxia tracers, such as [¹⁸F]FAZA (15% [11]), [¹⁸F]FETNIM (60% [15]), and [¹⁸F]HX4 (45% at 3.6 h [14]). In general, more hydrophilic compounds will have more rapid biodistribution and clearance from the body [16]. [¹⁸F]DiFA may thus be the ideal tracer for hypoxia imaging. In a comparison of our present dosimetry data with the available literature on [¹⁸F]FMISO [13], [¹⁸F]FAZA [11], [¹⁸F]HX4 [14], and [¹⁸F]FETNIM [15], radiation dose of these tracers resulted in almost the same distribution as that of [¹⁸F]DiFA. Although, in the case of [¹⁸F]DiFA, the radiation dose to the bladder wall is increased due to the high water solubility of this tracer, it is expected that the actual radiation dose can be reduced by shortening the urinary excretion interval (i.e., frequent voiding). No acute radiation toxicity was expected with 740 MBq F-18. Also, no chemical toxicity was expected with the unlabeled part of the 0.310 ± 0.109 μg DiFA since the maximum non-toxic dose was 517 μg/kg based on our preclinical study. We confirmed that there was no toxicity based on the laboratory tests.

To assess the potential of [¹⁸F]DiFA as a specific PET tracer for hypoxia imaging, we examined seven patients with malignant tumors by both [¹⁸F]FMISO and [¹⁸F]DiFA PET/CT imaging. In this study, there was a 48-h interval between [¹⁸F]FMISO and [¹⁸F]DiFA imaging. Hypoxia is an unstable condition that can be altered in a short time, theoretically explained by acute and chronic hypoxia. In our previous clinical study using [¹⁸F]FMISO, however, we confirmed that the degree and localization of hypoxia were stable between 2 scans of [¹⁸F]FMISO-PET with 48 h interval [17]. Therefore, we assumed that the hypoxia status was stable between [¹⁸F]FMISO and [¹⁸F]DiFA imaging. The results showed that there were no significant differences between [¹⁸F]FMISO and [¹⁸F]DiFA PET for the detection of tumor hypoxia. Moreover, [¹⁸F]DiFA PET at both 1 and 2 h post-injection showed better inter-observer reproducibility than [¹⁸F]FMISO at 4 h. Because [¹⁸F]FMISO has characteristics of slow accumulation and slow clearance from blood and normal tissues, background organs often show high accumulation when imaged, resulting in a low tumor-to-background ratio (Fig. 6). Early scans are greatly affected by perfusion and degrade accuracy and reproducibility. However, the image quality was poor in [¹⁸F]FMISO PET image 4 h after injection due to > 2× half-life of F-18. We think these are the main reasons for degraded inter-operator reproducibility for [¹⁸F]FMISO PET. In contrast, [¹⁸F]DiFA is rapidly cleared from the blood and normal tissues, enhancing lesion-to-background contrast. In addition, early scan timing keeps a high signal-to-noise ratio, which leads to good image quality. The rapid clearance of [¹⁸F]DiFA provided an easy evaluation and high reproducibility. From our results, 4 h [¹⁸F]FMISO PET has considered the reference standard in clinical and [¹⁸F]DiFA PET showed 3 false positives out of 10 lesions. The disparity between the two tracer datasets may be mainly due to the pharmacokinetics of the tracer itself, but also partly by the possible fluctuation of hypoxic status (so-called, acute hypoxia).

The tracer activity was determined by our preclinical study of dose exposure using rat. The activity of 740 MBq of [¹⁸F]DiFA was acceptable for the first-in-man study as maximum activity, and we confirmed its safety in healthy subjects and cancer patients. From this dataset, we will determine the optimal activity of [¹⁸F]DiFA. Wang et al. [18] and Thorwarth et al. [19] reported that the kinetic model of [¹⁸F]FMISO PET was the 3-compartment model. The pharmacokinetic model of [¹⁸F]DiFA is considered the same as for [¹⁸F]FMISO. In these tracers, although the injected activity of the [¹⁸F]DiFA was almost double that of [¹⁸F]FMISO, the lesion-to-background ratio did not change with increased activity (Additional file 3). [¹⁸F]DiFA has the advantage of early scan time for reducing patients waiting time, easy synthesis for robust results, and good reproducibility for the assessment of hypoxia status in multicenter trials. In the present investigation, the SUVmax values obtained with [¹⁸F]DiFA were lower than those obtained with [¹⁸F]FMISO, in agreement with a preclinical study by Yasui et al. [20].

The most important limitation of [¹⁸F]FMISO is its high lipophilicity, which causes slow tracer accumulation, slow plasma clearance, and low tumor-to-background contrast [2]. Our group showed that [¹⁸F]FMISO PET for hypoxia imaging achieved better quality at 4 h compared to 2 h, with a better lesion-to-background ratio [21], better differential diagnoses between glioblastoma and lower-grade gliomas at 4 h [22] than at 2 h [23], and high test-retest reproducibility of the tracer distribution at 4 h [17]. In this study, thus, we set the 4-h after injection of [¹⁸F]FMISO as the gold standard.

New-generation hypoxia tracers have been developed to improve hydrophilicity and accelerate the tracers’ clearance from normal oxygenated tissues [24]. A previous report using a single murine xenograft tumor model condition showed [¹⁸F]FMISO, [¹⁸F]FAZA, and [¹⁸F]HX4 demonstrated similar tumor distributions, and highest TBRs for [¹⁸F]FAZA and [¹⁸F]HX4 were obtained at 2 h p.i. and 3 h p.i., respectively. [¹⁸F]FMISO and [¹⁸F]DiFA did not show plateau formation and had better TBR at later time points [24, 25]. There is no clinical study which compared the uptake between [¹⁸F]FAZA and [¹⁸F]FMISO, while [¹⁸F]HX4 imaging in head and neck cancer patients at 1.5 h p.i. was found to have TMR properties similar to those of [¹⁸F]FMISO at 2 h p.i. Wei et al. reported that [¹⁸F]FMISO showed significantly higher uptake than [¹⁸F]FETNIM in tumor/non-tumor ratio in lung cancer [26]. More data have been accumulated for [¹⁸F]FAZA than for any other second-generation hypoxia tracers, but the reproducibility of the scans using [¹⁸F]FAZA has not been established. While a preclinical micro-PET analysis showed voxel-to-voxel reproducibility between two baseline scans performed 24 h apart, another preclinical report in an animal tumor model showed that [¹⁸F]FAZA uptake was less reproducible after 48 h, even without additional anticancer treatment [5]. There has been no report about the reproducibility of [¹⁸F]FAZA in a clinical trial. In our preclinical research using ex vivo autoradiography, the uptake of [¹⁸F]DiFA, formerly called HIC101, was shown to have a significant positive correlation with regions of pimonidazole distribution, indicating that [¹⁸F]DiFA was selectively accumulated in tumor hypoxic regions [20, 27]. Yasui et al. reported that the tumor-to-muscle ratios were significantly higher as early as at 1 h post-injection. In the current study, the images of 1 h p.i. of [¹⁸F]DiFA and the images of 4 h p.i. of [¹⁸F]FMISO were equivalent, indicating the potential to obtain an image with good contrast at 1 h after injection.

Masaki et al. [28] and Shimizu et al. [7] described the mechanisms of [¹⁸F]FMISO and [¹⁸F]DiFA. In brief, the glutathione-conjugated reductive metabolites of the tracer are present in the hypoxic regions of tumor tissues, suggesting that the tracer undergoes the glutathione conjugation reaction following reductive metabolism in hypoxic cells. The glutathione conjugate of reduced tracer was the major metabolite involved in the hypoxia-specific accumulation. In our preclinical study, while [¹⁸F]DiFA accumulated in hypoxic cells was higher than that of [¹⁸F]FMISO, [¹⁸F]DiFA was rapidly cleared from the blood [7]. When administered to mice, the tumor-to-blood and the tumor-to-muscle ratios of [¹⁸F]DiFA were higher than those of [¹⁸F]FMISO. The rapid clearance may have contributed to the reduced non-specific accumulation of [¹⁸F]DiFA in the background tissues. Therefore, the distribution of [¹⁸F]DiFA may be equally or highly specific to the hypoxic region of tumor tissues compared to that of [¹⁸F]FMISO [7]. In the current human study, the background of [¹⁸F]DiFA tended to be lower compared to that of [¹⁸F]FMISO (Additional file 4: Figure S1). Thus, [¹⁸F]DiFA achieved better contrast imaging of tumor hypoxia with a shorter waiting time compared to [¹⁸F]FMISO via the same mechanism.

The major limitations of this preliminary study were that only seven patients were enrolled for the diagnostic assessment of hypoxia detection by [¹⁸F]FMISO and [¹⁸F]DiFA and the primary tumors or metastases were varied, rather than restricted to a specific type of lesion. We plan to conduct verification studies on specific carcinomas soon. Since [¹⁸F]DiFA undergoes the same metabolic pathway of [¹⁸F]FMISO in hypoxic cells, the distribution of [¹⁸F]DiFA in tumor tissues might be affected by factors related to glutathione conjugation. Previously, our group investigated the mechanisms of the [¹⁸F]FMISO uptake in three cell lines (FaDu, LOVO, and T24) and found that [¹⁸F]FMISO accumulation was associated with glutathione conjugation ability as well as hypoxic conditions [29]. Similarly, [¹⁸F]DiFA accumulation might be affected by cellular factors such as the cellular reduced glutathione level and glutathione S-transferases, which catalyze glutathione conjugation reactions, and the multidrug resistant protein 1, which exports various kinds of glutathione conjugates out of cells. Therefore, further preclinical and clinical studies are needed to clarify whether these factors actually influence the imaging of [¹⁸F]DiFA. In addition, we studied whether [¹⁸F]FMISO and [¹⁸F]DiFA exhibit the same distribution 48 h apart, but we did not compare [¹⁸F]DiFA and new hypoxia tracers such as [¹⁸F]FAZA or [¹⁸F]HX4. Further research on the reproducibility of [¹⁸F]DiFA findings is also necessary.

The current study revealed 2 important findings. First, [¹⁸F]DiFA is well tolerated and its radiation dose is comparable to that of [¹⁸F]FMISO and other hypoxia tracers. [¹⁸F]DiFA showed very rapid clearance and a large fraction of renal excretion. Second, [¹⁸F]DiFA achieved an equivalent image quality compared with [¹⁸F]FMISO, with smaller inter-observer variability. Thus, [¹⁸F]DiFA PET enables hypoxia imaging with equivalent contrast in shorter waiting time and would be potentially suitable for a multicenter trial.