*Review* **Radiopharmaceutical Labelling for Lung Ventilation/Perfusion PET/CT Imaging: A Review of Production and Optimization Processes for Clinical Use**

**Frédérique Blanc-Béguin 1,\* , Simon Hennebicq <sup>1</sup> , Philippe Robin <sup>1</sup> , Raphaël Tripier <sup>2</sup> , Pierre-Yves Salaün <sup>1</sup> and Pierre-Yves Le Roux <sup>1</sup>**


**Abstract:** Lung ventilation/perfusion (V/Q) positron emission tomography-computed tomography (PET/CT) is a promising imaging modality for regional lung function assessment. The same carrier molecules as a conventional V/Q scan (i.e., carbon nanoparticles for ventilation and macro aggregated albumin particles for perfusion) are used, but they are labeled with gallium-68 (68Ga) instead of technetium-99m (99mTc). For both radiopharmaceuticals, various production processes have been proposed. This article discusses the challenges associated with the transition from 99mTcto <sup>68</sup>Ga-labelled radiopharmaceuticals. The various production and optimization processes for both radiopharmaceuticals are reviewed and discussed for optimal clinical use.

**Keywords:** V/Q PET/CT; [68Ga]Ga-MAA; <sup>68</sup>Ga-labelled carbon nanoparticles

#### **1. Introduction**

Lung ventilation-perfusion (V/Q) scintigraphy allows the regional lung function distribution of the two major components of gas exchanges, namely ventilation and perfusion, to be assessed [1]. Regional lung ventilation can be imaged after inhaling inert gases or radiolabelled aerosols that reach alveoli or terminal bronchioles. Regional lung perfusion can be assessed after intravenous injection of radiolabelled macroaggregated albumin (MAA) particles trapped during the first pass in the terminal pulmonary arterioles [2,3].

Pulmonary embolism (PE) diagnosis is the main clinical indication of lung V/Q scintigraphy in pulmonary embolism (PE) diagnosis. V/Q scanning was the first noninvasive test validated for PE diagnosis. The technique was then further improved with the introduction of single-photon emission computed tomography (SPECT) and, more recently, SPECT/computed tomography (CT) imaging [4]. There are many other clinical situations in which an accurate assessment of regional lung function may improve patient management besides PE diagnosis. This includes predicting post-operative pulmonary function in patients with lung cancer, radiotherapy planning to minimize the dose to the lung parenchyma with preserved function and reduce radiation-induced lung toxicities, or pre-surgical assessment of patients with severe emphysema undergoing a lung volume reduction surgery. However, although lung scintigraphy should play a central role in these clinical scenarios, its use has not been widely implemented in daily clinical practice [5]. One of the likely explanations could be the inherent technical limitations of SPECT imaging for the accurate delineation and quantification of regional ventilation and perfusion function [4].

Lung V/Q positron emission tomography (PET)/CT is a novel promising imaging modality for regional lung function assessment [6,7]. The technique has shown promising

**Citation:** Blanc-Béguin, F.; Hennebicq, S.; Robin, P.; Tripier, R.;

Salaün, P.-Y.; Le Roux, P.-Y. Radiopharmaceutical Labelling for Lung Ventilation/Perfusion PET/CT Imaging: A Review of Production and Optimization Processes for Clinical Use. *Pharmaceuticals* **2022**, *15*, 518. https://doi.org/10.3390/ ph15050518

Academic Editor: Klaus Kopka

Received: 16 March 2022 Accepted: 20 April 2022 Published: 22 April 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

results in various clinical scenarios, including PE diagnosis [8], radiotherapy planning [9], or pre-surgical evaluation of lung cancer patients [10]. Several large prospective clinical trials are underway, such as (NCT04179539, NCT03569072, NCT04942275, and NCT05103670). The rationale is simple [5]. PET/CT uses the same carrier molecules as conventional V/Q scanning, i.e., carbon nanoparticles for ventilation imaging and MAA particles for perfusion imaging. Similar physiological processes are therefore assessed with SPECT or PET imaging. However, carrier molecules are labelled with gallium-68 (68Ga) instead of technetium-99m (99mTc), allowing the acquisition of images with PET technology. PET has technical advantages compared with SPECT, including higher sensitivity, higher spatial and temporal resolution, superior quantitative capability and much greater access to respiratory-gated acquisition [11].

This article discusses the challenges associated with the switch from 99mTc- to <sup>68</sup>Galabelled V/Q radiopharmaceuticals. The various synthesis and optimization processes for both radiopharmaceuticals are reviewed and discussed, focusing on optimal clinical use.

#### **2. Challenges of the Transition from 99mTc- to <sup>68</sup>Ga-Labelled Radiopharmaceuticals for Lung Imaging**

As the chemical and physical properties of 99mTc and <sup>68</sup>Ga are different, the transition from V/Q scintigraphy to V/Q PET/CT implies some adaptation.

99mTc, a metallic radionuclide, is the most widely available isotope in diagnostic nuclear medicine. It is found in oxidation states −I to VII, but the technetium (Tc) complexes for medical applications are found mostly in oxidation state V [12]. 99mTc, which mainly decays (88%) with a half-life of 6.02 h by gamma emission (E<sup>γ</sup> = 140 keV) to the ground state technetium-99 (99Tc), is obtained as 99mTcO<sup>4</sup> <sup>−</sup> from a molybdenum-99 (99Mo)/99mTc generator commercially available and compatible with the requirements of Good Manufacturing Practices (GMP). The Tc(VII) in 99mTcO<sup>4</sup> − has to be reduced to a lower oxidation state to produce a 99mTc-stable peptide complex or a reactive intermediate complex [13]. Tc(V) forms 5- or 6-coordinate complexes, always in the presence of multiple bond cores with heteroatoms such as oxygen (O), nitrogen (N) or sulfur (S), among which the most common in radiopharmaceuticals are the oxotechnetium and the nitridotechnetium cores [12].

The prevalent gallium (Ga) oxidation state in aqueous solution is +3, forming several gallate anions as gallium hydroxides Ga(OH)<sup>4</sup> − at pH superior to 7. Ga(III) is a hard acid and is strongly bound to ligands featuring multiple anionic oxygen donor sites according to HSAB (hard-soft acid-base). Still, some cases have shown it to have a good affinity for thiolates [14]. Ga(III) ions can form four, five and six bonds, explaining all the possible salts or chelates. <sup>68</sup>Ga decays with a half-life of 67.71 min by positron emission (88.88%) and electron capture (11.11%) to the ground state zinc-68 (68Zn). It is obtained from a commercially available germanium-68 (68Ge)/68Ga generator, compatible with GMP requirements.

The first challenge of the switch from 99mTc- to <sup>68</sup>Ga-labelled radiopharmaceuticals for lung V/Q imaging is to maintain the pharmacological properties of V and Q tracers. Both MAA and carbon nanoparticles labelled with 99mTc have been largely studied. They have been shown to have a biodistribution throughout the lungs that allow an accurate assessment of regional lung perfusion and ventilation function. The principle of lung V/Q PET/CT imaging is to assess similar physiological processes than with conventional V/Q scan, but with greater technology for image acquisition.

The technique needs to be easy to implement in nuclear medicine facilities to enable routine use. The preparation should be fast, simple, GMP-compliant and safe for the operators. Furthermore, radiopharmaceutical production should use unmodified commercially available kits of carrier molecules and similar equipment and devices as much as possible as those used for conventional V/Q scans.

#### **3. Lung Perfusion Imaging**

## *3.1. [99mTc]Tc-MAA*

## 3.1.1. Chemical Aspects of [99mTc]Tc-MAA Particles

Among the various type of human serum albumin (HSA) available for radionuclide labeling, MAA is the most commonly used form in nuclear medicine facilities. The nature of the complex [99mTc]Tc-MAA has not been fully elucidated. It was hypothesized that the labelling of proteins with 99mTcO<sup>4</sup> − involved reduction of the anionic Tc(VII) to a cationic Tc by the tin Sn(II) contained in the commercial kit, which was then complexed with electron-donating groups [15–17]. Some authors have assumed that 99mTcO<sup>4</sup> − reduced by the Sn(II)- albumin aggregates probably formed a (Tc = O)3+ complex with the aggregates [15]. More recently, high positive cooperativity was shown between 99mTc and MAA, although MAA particles did not seem to have binding pockets [18,19]. Moreover, it has been shown that the speed of radiolabelling increased from HAS to albumin nanocolloids (NC) to MAA due to the greater reaction surface [18]. This result agreed with the hypothesis, which assumed that in HAS labelling kits, Sn2+ may be enclosed in the tertiary structure of the protein and that it may take some time for the 99mTcO<sup>4</sup> <sup>−</sup> added to diffuse the site of Sn2+ for reduction reaction [17]. Furthermore, MAA has very complex shapes with larger surfaces than a spherical shape (as for NC) of equivalent diameter, enhancing the reactivity properties [18,19].

Whatever the exact nature of the link between 99mTc and MAA, the complex [99mTc]Tc-MAA demonstrates high stability as more than 90% of the radioactivity is still associated with the MAA after 24h of in vitro incubation in whole human blood at 37 ◦C [20].

## 3.1.2. Technical Aspects: [99mTc]Tc-MAA Preparation

[ 99mTc]Tc-MAA particles are manually prepared by introducing a 99mTc solution in a commercially available MAA kit. The 99mTc is obtained from a <sup>99</sup>Mo/99mTc generator as sodium pertechnetate (99mTcO<sup>4</sup> <sup>−</sup>, Na<sup>+</sup> ). The MAA labelling with 99mTc, which occurred at pH 6, is a simple and fast (about 15 min) process, which allows the production of GMP [ 99mTc]Tc-MAA without heating step [18].

Before intravenous administration to the patients, the [99mTc]Tc-MAA suspensions are tested according to the standards mentioned by the kit supplier for clinical use. The radiochemical purity (RCP) is generally controlled using instant thin layer chromatography (iTLC), and the radioactivity distribution is assessed by filtration of the [99mTc]Tc-MAA suspension through a 3-µm pore size membrane. The results are obtained by measuring filter and filtrate radioactivity. The radionuclidic purity and the pH have to be controlled as well. As MAA are large particles, [99mTc], Tc-MAA must be resuspended by gentle agitation before dispensing.

#### 3.1.3. Pharmacological Aspects

In a [99mTc]Tc-MAA suspension, the average particle size is 20–40 µm, and 90% have a size between 10 and 90 µm. There should be no particles larger than 150 µm [21]. [99mTc]Tc-MAA particles reach the lung via the pulmonary arterial circulation. Due to the size of the alveolar capillaries (5.5 µm on average), the [99mTc]Tc-MAA does not reach the alveolar capillaries but largely accumulates in the terminal pulmonary arterioles. Particles inferior to 10 µm may pass through the lungs and then phagocytose by the reticuloendothelial system [21]. According to the requirement of the MAA suppliers, the number of MAA particles injected should range from 60,000 to 700,000 to obtain uniform distribution of activity reflecting regional perfusion (for over 280 billion pulmonary capillaries and 300 million pre-capillary arterioles) [22].

Many studies have shown the suitability of the [99mTc]Tc-MAA suspension to perform pulmonary perfusion scintigraphy. In rabbits, it has been shown that more than 90% of the activity was found in the lungs within a few minutes of administration and that greater than 80% of the activity remained in the lungs over the first hour of the study [20]. Malone et al. assessed the biodistribution of MAA particles in humans [23]. A total of 98% of

activity was measured in the lungs immediately after injection. The removal of activity from the lungs followed an approximately bi-exponential form with the first phase in which 56% of the components had an effective half-life of 0.88 ± 0.16 h and the second phase in which 44% of the components had a half-life of 4.56 ± 0.39 h. Moreover, 3 h after injection, the [99mTc]Tc-MAA uptake in the kidneys and the bladder was 3.6 <sup>±</sup> 2.1% and 5.1 <sup>±</sup> 4.0%, respectively [23].

## *3.2. [68Ga]Ga-MAA*

## 3.2.1. Chemical Aspects of [68Ga]Ga-MAA Particles

MAA labelling with <sup>68</sup>Ga has been proposed using bifunctional chelators such as EDTA or DTPA, forming quite stable and inert chelates [24,25]. However, direct labelling was performed by most groups. Direct labelling uses a co-precipitation of <sup>68</sup>Ga(III) and albumin particles [26,27]. Mathias et al. hypothesized that <sup>68</sup>Ga was adsorbed to the surface of the MAA particles after hydrolysis to insoluble gallium hydroxide without excluding specific interactions of Ga(III) ion with ion pairs exposed at the particle surface [28]. As Ga is present as Ga(OH)<sup>4</sup> <sup>−</sup> at a basic pH, <sup>68</sup>Ga does not bind to MAA at a pH above 7. The MAA behavior matches with solvent-exposed glutamate and aspartate amino acids, which should be binding sites for multivalent cations with low affinity and low cation specificity [18]. Furthermore, Jain et al. found that stannous chloride (SnCl2) present in the MAA kits for the reduction of 99mTc had a strong influence on [68Ga]Ga-MAA formation (radiochemical yield, mean particle diameter, serum stability), suggesting that Sn could be linked to MAA or <sup>68</sup>Ga [29]. More recently, it has been assumed that MAA has multiple affinity binding sites for <sup>68</sup>Ga [18]. Moreover, during 99mTc and <sup>68</sup>Ga competition evaluation for MAA binding sites, MAA showed no discrimination between 99mTc and <sup>68</sup>Ga coherently without a binding pocket [18].

## 3.2.2. Technical Aspects: [68Ga]Ga-MAA Preparation

[ 99mTc]Tc-MAA preparation is a manual and simple process involving only 2 steps: generator elution and mixing the eluate with the MAA. In contrast, because of the chemical properties of <sup>68</sup>Ga, at least four steps are required to label MAA particles with <sup>68</sup>Ga: <sup>68</sup>Ge/68Ga generator elution, mixing the <sup>68</sup>Ga eluate with the MAA, heating the reaction medium and the purification of the [68Ga]Ga-MAA. The key steps of MAA labelling with <sup>68</sup>Ga are presented in Figure 1. *Pharmaceuticals* **2022**, *15*, 518 7 of 18

**Figure 1.** Key points of MAA labelling with 68Ga. **Figure 1.** Key points of MAA labelling with <sup>68</sup>Ga.

• 68Ga eluate

• MAA As shown in Table 1, almost all authors used commercial kits available to prepare [99mTc]Tc-MAA for labelling MAA particles with 68Ga. The use of commercially available kits is an important consideration to facilitate the implementation of the technique in nuclear medicine facilities. However, commercially available MAA kits contain SnCl2 and Table 1 summarizes the various [68Ga]Ga-MAA preparation processes described in the literature. The main diverging points of the preparations include (1) the choice of MAA particles, (2) the need for a <sup>68</sup>Ga eluate pre-purification, (3) the labelling conditions (pH, heating temperature and time), (4) the [68Ga]Ga-MAA suspension purification, and (5) the automation of the process (Figure 1).

free albumin. Consequently, many groups carried out MAA labelling with washed MAA to remove the excess of free albumin and SnCl2 (stannous chloride), which is usually used

Ayşe et al. obtained a better final product RCP by washing the MAA particles before the labelling (RCP = 99.0 ± 0.1%) rather than not washing (RCP = 95.0 ± 0.1%) [38]. On the other hand, Mueller et al. found no significant difference in the radiolabelling yields using non-washed and pre-washed MAA (80% and 75%, respectively) [35]. In studies that used unmodified commercially available MAA kits, radiolabelling yields were consistently superior to 75.0% (Table 1) [28,35,39]. Furthermore, Jain et al. found lower radiolabelling yields using in-house synthesized MAA without SnCl2 than MAA with SnCl2 (49.9 ± 1.3% and 84.5 ± 5.3%, respectively). They found that stannous chloride present in the MAA kits used had a strong influence on the [68Ga]Ga-MAA formation (radiochemical yield, mean particle diameter, serum stability), suggesting that Sn could be linked to MAA or 68Ga [29]. Consequently, using unmodified non-washed commercially available MAA kits pro-

68Ga eluate obtained from currently available generators are contaminated with longlived parent nuclide 68Ge and cationic metal ion impurities such as titanium (Ti)4+ from the column material, zinc (Zn)2+ from the decay of 68Ga or iron (Fe)3+. These impurities might

Pre-purification of the eluate has been proposed by several groups, with various methods such as anion exchange chromatography, cationic cartridge, fractionation or eluate pre-concentration (Table 1) proposed to overcome this issue. Most groups performed an eluate pre-purification using an SCX cartridge or an equivalent pre-conditioned with

In contrast, a few groups did not perform 68Ga eluate pre-purification before MAA labelling and obtained 68Ge impurity levels lower than 0.0001% and radiolabelling yields superior to 96.0% (Table 1) [28,29,34,39]. Based on these results and the fact that this is time-consuming, the pre-purification of the 68Ga eluate does not seem mandatory and

duced for 99mTc seems to be a suitable solution for [68Ga]Ga-MAA labelling;

compete with 68Ga in the complexation reaction [40,41].

hydrochloric acid (HCl) and water (Table 1).

could be avoided for [68Ga]Ga-MAA preparation.

• Labelling conditions




*Pharmaceuticals* **2022**, *15*, 518

**Table 1.** *Cont.*

