Dealing with PET radiometabolites

Positron emission tomography (PET) is one of the most rapidly progressing imaging modalities, which offers understanding about the biochemical, physiological, and pharmacological functions at a molecular and cellular level [1, 2]. To date, PET has been applied in several areas including cardiology, neurology, and oncology particularly in tumor targeting and diagnosis [3, 4]. Numerous PET radiotracers have been studied and developed for imaging cancerous lesions, brain receptors, transporters, and enzyme systems, but only a few of them are currently in use as imaging agents in clinical practice. The PET imaging technique includes the acquirement of physiological images that constitute the distribution of radioactivity associated with positron-emitting radionuclides in the volume of examination [5]. Compared to other imaging tools like the X-ray, magnetic resonance imaging (MRI), and computed tomography (CT) which offer structural information, PET imaging is advantageous as it shows numerous functional and biochemical characteristics of organs and tissues of interest. PET combines several crucial features like absolute quantitation, allows repetitive and longitudinal examination before/after interventions with the help of short-lived PET radionuclides. Very high resolution (< 2.5 mm) can be achieved at any part of the body with a dedicated PET scanner [6, 7]. Most importantly, procedures developed in preclinical research can be applied to clinical studies. The PET imaging technique is currently the only non-invasive imaging tool to measure in vivo tissue pharmacokinetics quantitatively.

The process involved in developing new PET radiopharmaceuticals begins with compound selection, through in vitro and in vivo efficacy evaluation, ultimately leading to clinical investigation. Hence, the performance of a PET study mostly depends on the used radiotracer of interest. However, developing PET imaging tracers is difficult as various critical criteria need to be fulfilled such as optimum molecular weight and lipophilicity. Besides, the radiotracer should have a high affinity for the target receptor, and it should show a fast washout after reaching the saturation binding. For imaging the central nervous system (CNS), the radiotracer should not be a subject for P-glycoprotein (P-gp) [8‐10]. On the other hand, the non-brain tissue radiotracer development process also faces some serious challenges as low peripheral radiometabolism is necessary to compensate for the lack of BBB. In the case of the CNS radiotracer development, most of the compounds failed to show promising results in preclinical and clinical studies. Some of the most common causes for the failure of radiotracers appear when non-displaceable binding surpasses target binding, i.e., when signals from the background exceed the signals coming from the target. One of the serious problems with PET is that it offers information about the total radioactivity deposited in the tissue of interest but does not reflect any evidence of its chemical identity. The existence of radiometabolites in the tissue of interest would affect how precise the quantitative analysis of the PET examination is [11, 12].

In living organisms, PET radiotracers usually encounter different types of chemical modifications including oxidation, reduction, hydrolysis, decarboxylation, and various conjugation processes. Due to these biotransformations, diverse chemical entities are generated while the concentration of the original radiotracer decreases [13‐18]. Therefore, the signal measured by the PET scanner replicates the entire amount of radioactivity deposited in the tissue of choice, but it does not offer any indication about the chemical identity of the pharmacophore itself. From a radiopharmaceutical perspective, the quantification of only the parent radiotracer fraction present in tissues is a requirement [19]. Almost all the PET radiotracers are metabolized within the body. Among these metabolized products, some may carry the radioactivity tag. There are some reactions that are planned, such as the phosphorylation of [¹⁸F]FDG and its resulting trapping into the cell. However, the PET radiotracers are mostly metabolized to form unwanted radiometabolites which create serious problems in quantification. Although radiometabolites present in plasma can be rectified, the radiometabolites deposited into tissues should be considered during image analysis. Radiotracers for brain imaging are designed in such a way that they do not produce any radiometabolites that can pass through the blood–brain barrier [20]. However, there are some exemptions to this guideline, as in the case of [¹⁸F]FDOPA. In the preclinical evaluation of a new PET radiotracer, it is important to take into consideration that the radiometabolites may display a significant difference in distribution patterns and activity in the body in comparison to the parent radiotracer. Enough information on biochemical transformations is crucial to have a more detailed knowledge of radioactive distribution in the whole body. For example, substantial inter-tissue variances in the further radiometabolism of FDG, i.e., away from FDG-6-phosphate, have been demonstrated ex vivo in different rat organs [21]. Moreover, a relative tissue-dependent distribution of individual radiometabolites replicating the physiological features of the respective organs has also been established [21]. Therefore, by understanding the occurrence and biochemical nature of different radiometabolic compounds, it is possible to gain extensive insight into the pathological and physiological processes by PET. To calculate physiological parameters such as receptor density and metabolic rates, it is necessary to know the concentration of the tracer and its radiometabolites in the blood and tissues over time. Hence, it is vital to establish methods by which radiometabolites, especially those having the potential to cross the BBB, can be identified and measured over time. Identification of radiometabolites during the evolution of a new radiotracer is also important as it offers information about the most suitable position to radiolabel a compound to escape any production of inquisitive radiometabolites [22, 23]. There are some reviews already published on PET radiometabolites [24, 25]. However, those reviews mainly focus on the sample preparation techniques and recently available technology to improve the radiometabolite analysis process. Our review mainly summarizes the chemical and structural identities of radiometabolites of different radiotracers. Moreover, this review also highlights how a slight chemical modification could reduce the formation of radiometabolites, which could interfere with the results of PET imaging.

Radiometabolite adjusted arterial input function is crucial for the exact quantification of the PET radiotracers that do not possess any appropriate reference region in the brain. There are two main reasons for the chemical identification of radiometabolites: firstly, to determine the amount of parent radiotracers in plasma, and secondly, to rule out or correct any radiometabolite accumulation in peripheral tissue. Radiometabolite formations of the tracer might be a concern for the PET study, as radiometabolic products may display considerably contrasting administration patterns into the body, as compared to the tracer itself. An understanding of radiometabolite formation and its chemical nature may provide important information on physiological, biochemical, and pathological processes by PET. To fulfill this requirement, data analysis models were established to evaluate the correlation between the acquired data and the biophysical factors influencing radiotracer accumulation and biotransformation [26, 27]. Moreover, radiotracer metabolism can significantly influence the arterial input function, which is a factor applied for analyzing PET images quantitatively. Radiometabolite examinations in the plasma are a crucial requirement for the precise assessment and revelation of PET imaging and the determination of pathological changes in metabolic pathways [28]. After estimating the radioisotopic decay and restructuring it into an image, the exact biodistribution of the compound is accessible. Hence, radiometabolite analysis is an extremely important topic in PET imaging. In some cases, it was noticed that a radiometabolite can show a better affinity for the target than its parent radiotracer. For example, [¹¹C]Loperamide and its putative radiometabolite [N-methyl-¹¹C]N-desmethyl-loperamide both behave as the avid substrate for P-gp [29, 30]. Actually, [N-methyl-¹¹C]N-desmethyl-loperamide could be superior to [¹¹C]Loperamide in determining P-gp function at the blood–brain barrier because further demethylation of [N-methyl-¹¹C]N-desmethyl-loperamide will generate a more polar radiometabolites that have negligible access into the brain [31]. There are some instances where the parent radiotracer and its radiometabolites both have the affinity for the same targets. In this case, the cold compound of the radiotracer can be injected for checking their competition for the target. For example, both [¹⁸F]LBT-999 and its radiometabolite (2β-carbomethoxy-3b-(4′-tolyl) nortropane) have the binding affinity for dopamine transporter (DAT) [32]. In a PET experiment, Saba et al. have shown that after intravenous injection of cold metabolite, it induced a strong displacement of [¹⁸F]LBT-999 in the striata, suggesting that the radiometabolite can cross the BBB and competing with the radiotracer [¹⁸F]LBT-999 for the target [33].

Most of the drug/tracer metabolism takes place in the liver. However, it may also take place in the kidney or intestine. Usually, metabolism occurs to transform the radiotracer into a compound that is more water-soluble and easily excreted. Similar to the drug, the radiotracer metabolism also takes place in two phases. In phase I, the radiotracers are chemically changed by enzymes through oxidation, reduction, and hydrolysis. Hence, in phase I, a new functional group is generated which can be used in the next phase. In phase II, some hydrophilic moiety is attached to that functional group to form readily water-soluble conjugates that can be easily excreted through urine [34, 35]. Table 1 summarizes the different processes, substrates, and products of phase I and phase II.

Several chromatographic techniques including thin-layer chromatography (TLC) [36], high-performance liquid chromatography (HPLC) [37], micellar chromatography [38], and overpressure thin-layer chromatography (OPTLC) [39] were commonly used for the examination of radiometabolites. Among these techniques, TLC was the earliest known chromatographic tool applied in radiochemistry to separate radioactive compounds. To date, TLC is an important technique and is regularly used in radiopharmacy departments, to determine the radiochemical purity of radiopharmaceuticals. This technique is very easy to apply, fast, inexpensive, and most importantly it can detect all deposited radioactivity without losing any information [40]. However, TLC, generally a qualitative technique, suffers from several limitations including resolution, accuracy, and reproducibility [41]. Therefore, TLC is not a suitable tool for radiometabolite analysis. In comparison to TLC, the OPTLC comes with superior resolution and rapid analysis. Until recently, the most widely used technique for the analysis of radiometabolites of a radiotracer is the HPLC, combined with radioactivity detectors. HPLC analysis is reproducible, and it allows to quantify narrow, rider, and shoulder peaks precisely which is not possible for TLC. A multistep sample extraction process is required before it could be injected into the HPLC system [42]. However, due to the reasonably lengthy preparation procedure in combination with the very short half-lives of the radiotracers, loss of information may occur particularly at the later stages of the PET imaging experiments. Additionally, multi-stage blood extraction may lead to a decrease or alteration of information. This may induce bias and hamper the repeatability of the analysis. To get rid of this time-consuming sample preparation technique, solid phase extraction (SPE) of the radiometabolite can serve as a suitable choice. In SPE, first, the plasma sample is loaded into an SPE cartridge directly or after the precipitation of protein. Then the radiotracer and its metabolites are extracted from SPE depending on their polarity. Although direct blood sampling could largely reduce the sample preparation time, it is still a challenging issue in radiometabolite analysis. Another important and well-known approach for radiometabolite analysis is column-switching HPLC. Here, the plasma sample is directly loaded into a capture column which is a kind of short SPE cartridge. Following this, the radiotracer and its metabolites are eluted into a reverse-phase column of a radio-HPLC [43‐45]. This semi-automated method effectively reduces time-consuming sample preparation steps and exposure to the radioactivity with better reproducibility. However, this method suffers from high back pressure as it is required a large plasma volume for radiometabolite analysis. Nakao et al. have developed an alternative rapid and efficient method based on micellar liquid chromatography for the direct analysis of plasma radiometabolites [24, 38, 46]. This method uses micellar/high submicellar liquid chromatography along with semi-preparative alkyl-bonded silica column for the analysis of radiometabolites. The steep gradient and high flow rate are used for the rapid removal of the macromolecule from plasma. Compared to the particle-based column, the monolithic column provides high resolution and better sensitivity. The main advantage of this approach is that it reduces time-consuming sample preparation steps. Besides, it also allows the analysis of a large number of samples with appropriate sensitivity. Currently, protein precipitation and SPE of radiometabolites are the two most encouraging sample preparation procedures designed for radioactive metabolite analysis. Table 2 summarizes the various advantages and limitations of different analytical techniques applied to radiometabolite analysis.

The most commonly used biological samples for the radiometabolite analysis are blood, plasma, and tissue. The direct analysis of these samples has several limitations. Hence, most of the time, it is essential to separate the radiotracer and its metabolites from the biological matrix.

One of the most important aspects of PET study is to find out the amount of free fraction of an injected radiotracer in plasma. After the injection of the radiotracer into the body, a fraction of it binds to the plasma protein. The remaining fraction (free radiotracer) is then making an equilibrium with its plasma protein bounded fraction [42]. It is worth mentioning that only the free fraction of the radiotracer is available to bind the target of interest and the efficacy of a drug depends on the free fraction present in plasma [47]. When the two drugs were administrated into the body at the same time, it is particularly important to know their plasma protein binding affinity as one drug can replace a proportion of other drugs from being bound to the plasma protein, enabling a greater proportion of free drug fraction. The methods to measure the amount of plasma protein binding and the free fraction of radiotracer were discussed in detail by Moein et al [25].

Improvement of the metabolic stability of the radiotracer is as important as the analysis and identification of its radiometabolites. An increase in metabolic stability could improve the quality of PET imaging results. There are two major ways to improve the metabolic stability of the radiotracer. The first approach is to substitute the hydrogen present in the reactive site by deuterium. The deuterium substitution of the methyl or ethyl group will usually reduce the rate of metabolism as it requires higher energy to cleave a carbon–deuterium covalent bond other than the carbon–hydrogen bond simply due to a higher mass effect of deuterium. The second approach is to change the labeling position without altering any of the structural or chemical changes of the parent radiotracer. In this section, we will look into the case study for the above two approaches using [¹⁸F]Fluorocholine and [¹¹C]WAY-100635, respectively.

PET is presently one of the most beneficial imaging tools for measuring drug pharmacokinetics non-invasively in the tissue of interest. Since the last decade, PET studies have offered a lot of important information regarding the tissue accumulation of numerous types of drugs. However, for the analysis and interpretation of the images, it is required to keep in mind that the collected signals from the PET scanner alone do not permit any conclusion to be drawn about the compound’s chemical identity that contributes to the radioactivity during the scan. The identification of radiometabolites is extremely important as they could offer some important information about the appropriate part of the compound to attach the radioactive label. From the image analysis point of view, detailed knowledge of the radiometabolite formation is required to get the maximum information from a PET scan. However, the identification of the radiometabolites is quite difficult because of its very short-lived radioisotope and the low dosage of the radiopharmaceutical and its metabolites. The tedious sample preparation procedure makes it even more complicated to analyze the radiometabolite in in vivo. Here, in this review, we have given important information and updates regarding the radiometabolite analysis of several carbon-11- and fluorine-18-based radiotracers from in vitro, preclinical and clinical aspects.