*Article* **Kinetic Modelling of [68Ga]Ga-DOTA-Siglec-9 in Porcine Osteomyelitis and Soft Tissue Infections**

**Lars Jødal 1,\*, Anne Roivainen 2,3, Vesa Oikonen 2,3, Sirpa Jalkanen 4, Søren B. Hansen 5, Pia Afzelius 6, Aage K. O. Alstrup 5, Ole L. Nielsen <sup>7</sup> and Svend B. Jensen 1,8**


Academic Editor: Derek J. McPhee

Received: 2 October 2019; Accepted: 10 November 2019; Published: 13 November 2019

**Abstract:** Background: [68Ga]Ga-DOTA-Siglec-9 is a positron emission tomography (PET) radioligand for vascular adhesion protein 1 (VAP-1), a protein involved in leukocyte trafficking. The tracer facilitates the imaging of inflammation and infection. Here, we studied the pharmacokinetic modelling of [68Ga]Ga-DOTA-Siglec-9 in osteomyelitis and soft tissue infections in pigs. Methods: Eight pigs with osteomyelitis and soft tissue infections in the right hind limb were dynamically PET scanned for 60 min along with arterial blood sampling. The fraction of radioactivity in the blood accounted for by the parent tracer was evaluated with radio-high-performance liquid chromatography. Oneand two-tissue compartment models were used for pharmacokinetic evaluation. Post-mortem soft tissue samples from one pig were analysed with anti-VAP-1 immunofluorescence. In each analysis, the animal's non-infected left hind limb was used as a control. Results: Tracer uptake was elevated in soft tissue infections but remained low in osteomyelitis. The kinetics of [68Ga]Ga-DOTA-Siglec-9 followed a reversible 2-tissue compartment model. The tracer metabolized quickly; however, taking this into account, produced more ambiguous results. Infected soft tissue samples showed endothelial cell surface expression of the Siglec-9 receptor VAP-1. Conclusion: The kinetics of [ 68Ga]Ga-DOTA-Siglec-9 uptake in porcine soft tissue infections are best described by the 2-tissue compartment model.

**Keywords:** kinetic analysis; Siglec-9; gallium-68; vascular adhesion protein; VAP-1; infection; inflammation; osteomyelitis; animal model; *Staphylococcus aureus*

#### **1. Introduction**

Infections causing localized lesions in the body, e.g., osteomyelitis, cannot always be successfully treated by the systemic administration of antibiotics; instead, surgical intervention may be necessary, which requires knowledge of the site(s) of infection. Assuming the availability of a suitable tracer, positron emission tomography (PET) is well suited for the task. However, it has proven difficult to find a tracer with both high and specific uptake in infected tissue.

The current standard tracer used for PET imaging of infectious and inflammatory diseases is the fluorine-18 labelled glucose analogue 2-deoxy-2-[18F]-fluoro-d-glucose ([18F]FDG) [1]. Generally, [ 18F]FDG shows high uptake in sites of infection and inflammation, but the uptake is not specific: [ 18F]FDG accumulates in all cells that use glucose as an energy source, including not only activated immune cells but also rapidly proliferating cancer cells, brain tissue and working muscle tissue.

Leukocytes (white blood cells, WBC) are natural seekers of infection. Leukocytes labelled with 111In or 99mTc have been widely used for scintigraphy imaging of infection, but the labelling procedure is time consuming and requires withdrawing 30–40 mL of blood from the patient [2]. A simpler alternative is anti-granulocyte monoclonal antibodies (anti-G-mAbs) labelled with 99mTc. However, while the isotopes 111In and 99mTc are suitable for gamma camera imaging, they cannot be used for PET imaging. This is a technical disadvantage, as the counting efficiency is typically an order of magnitude higher for the PET scanner than for the gamma camera, and the spatial resolution is superior. Labelling of leukocytes with [18F]FDG for PET imaging has been investigated, but the labelling efficiency is lower, and [18F]FDG elutes from the leukocytes over time [3]. Furthermore, the molar activity of labelled leukocytes will be many orders of magnitude higher than in the patient as a whole, for which reason the short-range β<sup>+</sup> radiation (positrons before annihilation) can result in damaging self-irradiation to [ 18F]FDG-labelled leukocytes [4].

The relationship between time and physical decay must also be noted. Injected leukocytes take hours or more to accumulate at the infection site, and imaging can be performed 3–4 h after injection at the earliest [2]. These delays are acceptable with 111In (T1 2 = 2.8 days) and 99mTc (T1 2 = 6 h), but in relation to PET imaging, the shorter physical half-lives of the two most widely used isotopes, 18F (T1 = 110 min) and 68Ga (T1 = 68 min), make long uptake times problematic.

2 2 Instead of labelled leukocytes, a tracer directly related to the processes causing leukocyte accumulation in infected tissues is being sought. Such a tracer would potentially enable faster imaging, and at the same time, the handling of blood products from the patient would be avoided. A relevant target molecule is vascular adhesion protein 1 (VAP-1), currently known as a primary amine oxidase (AOC3, EC 1.4.3.21), which is acting both as an adhesion molecule and as a regulatory enzyme in the process of leukocyte binding to the endothelium of blood vessels in infected and inflamed tissue [5,6]. Under normal conditions, VAP-1 is stored in intracellular granules. Upon an inflammatory stimulus, VAP-1 is rapidly translocated to the endothelial cell surface, where it is readily accessible to intravenously administered PET ligands.

As reviewed in [7], 68Ga-labelled synthetic peptides that bind to VAP-1 have been investigated in different experimental infection and inflammation models, and some have shown a better distinction than [18F]FDG between cancer and inflammation. Overall, the review considers VAP-1 to be an optimal target for imaging of inflammation. One of the reviewed tracers, [68Ga]Ga-DOTA-Siglec-9, is currently in a phase I clinical trial (trial no. NCT03755245).

It may be noted that VAP-1 imaging will not distinguish between infection and inflammation, but because VAP-1 is involved in leukocyte extravasation, it is directly linked to the body's natural response to infection. Imaging of VAP-1 expression may even be used to study this process, especially if the kinetics of the VAP-1 imaging tracer are known.

Sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) is a natural ligand for VAP-1 and is expressed on monocytes and neutrophils [8,9]. The PET tracer [68Ga]Ga-DOTA-Siglec-9 contains a modified fraction of the full Siglec-9 protein and has been found to accumulate in infected tissues [10,11] as well as inflammation sites [8,11–13] and certain tumours [8]. However, the kinetics of [ 68Ga]Ga-DOTA-Siglec-9 have been investigated only in a single study that addressed inflammation [12]; thus studies on the kinetics of this tracer in infections are lacking.

In earlier studies, we investigated the PET imaging of infection in a porcine osteomyelitis model. In this model, *Staphylococcus aureus* (*S. aureus*) bacteria were injected into the right femoral artery of juvenile pigs to haematogenously induce osteomyelitis in the right hind limb without trauma to the bone [14–16]. In a number of animals, both osteomyelitis and soft tissue infections were induced in the limb, allowing us to compare the uptake in these two types of tissues. Each animal was scanned with a series of radioactive tracers within the same day, with planned delays to allow one tracer to physically decay before the next was injected [17].

The present study investigates the kinetics of [68Ga]Ga-DOTA-Siglec-9 in this porcine osteomyelitis model, including a comparison of the uptake in osteomyelitis and in soft tissue infections.

#### **2. Results**

The characteristics of the eight pigs in the study are summarized in Table 1.


**Table 1.** Characteristics of the pigs in the study.

\* The numbering follows the order in the overall osteomyelitis project described in the main text. Pigs no. 6, 9, and 10 have been described regarding other tracers in previous publications: [18,19] (same numbers) and [20] (where A–E corresponds to numbers 6–10). All pigs in the table except no. 24 were also included in [21] (with different numbering). Blood sample data on [68Ga]Ga-DOTA-Siglec-9 in pig no. 10 have been described in [22]. † Pig no. 26 was scanned twice with [68Ga]Ga-DOTA-Siglec-9. The second scan was performed 4.6 h (approximately 4 physical half-lives of 68Ga) after the first scan and with a higher level of injected radioactivity. Prior to the second scan, 5 mg of unlabelled ("cold") DOTA-Siglec-9 peptide in 10 mL had been slowly injected intravenously (i.v.) into the pig to block receptors—see main text.

## *2.1. Visual Uptake*

Visually, [68Ga]Ga-DOTA-Siglec-9 showed elevated uptake in inflamed and infected soft tissues, while not being prominent in infected bone (osteomyelitis). As an example, a coronal view of the uptake around the femur in pig no. 25 is shown in Figure 1.

At late time points, the decay-corrected PET signal was markedly reduced in all tissues outside the bladder, indicating that uptake of the tracer is reversible rather than irreversible (see Figure 2).

**Figure 1.** Representative PET/CT image from pig no. 25. The pig was lying supine with roughly upward-pointing limbs, and the view is coronal, seen from the ventral side of the animal ("front" = opposite of back is in the direction of the reader); the top of the image is in the direction of the animal's head. Full arrow: Uptake in phlegmon/early abscess at the right distal femur. Dotted arrow: Similar non-infected tissue for comparison. Red circles show sections of volumes of interest (VOIs) in soft tissue, black ellipse/circle show VOIs in bone tissue. The PET image represents the summed data from 15 to 30 min post-injection (p.i.). The tissue samples presented in Figure 3 were taken from positions approximately corresponding to the soft tissue VOIs.

**Figure 2.** Late PET/CT image from pig no. 25. Static scan with [68Ga]Ga-DOTA-Siglec-9 at approximately 70 min p.i.; position and colour scale correspond to those in Figure 1 (radioactivity is corrected for physical decay).

#### *2.2. VAP-1 Expression Studied by Immunofluorescence (IF)*

To analyse the expression of VAP-1 in infectious/inflammatory foci and to determine whether VAP-1 is translocated from intracellular granules to the endothelial cell surface, pig no. 25 was administered anti-VAP-1 antibody shortly before euthanasia, as described in the Methods section. The tissue samples were stained either with anti-VAP-1 + secondary antibody or with secondary antibody alone. Together, this pair of stainings allowed us to detect both the total VAP-1 pool and the part of the pool reached by the intravenously injected antibody; presumably only the latter pool is accessible to the imaging peptide. It can be noted that even though Siglec-9 and anti-VAP-1 both bind to VAP-1, their binding sites are different [9]; thus, they do not compete for binding.

VAP-1 was highly expressed in abscess-associated vasculature and a portion of this pool was detected with the intravenously injected antibody (Figure 3A,B). While VAP-1 is intracellularly expressed in certain vessels of normal soft tissue, no signal was detected with the intravenously administered antibody (Figure 3C,D). It thus appears that VAP-1 is accessible to blood-circulating antibodies only in the infected/inflammatory tissue, which can be seen as an indication that the translocation of VAP-1 to the cell surface is inflammation-induced.

**Figure 3.** IF staining of infected and non-infected tissue. Stained samples from pig no. 25. **A** and **B** are stains of serial sections from phlegmon/early abscess at the right distal femur. For comparison, **C** and **D** are stains of a non-infected soft tissue sample from an anatomically corresponding position in the left limb of the same animal. (**A**) Staining with anti-VAP-1 and the secondary antibody showed high intracellular and surface expression of VAP-1 in vessels. The scale bar is 50 μm. (**B**) Staining only with the secondary antibody showed VAP-1 surface expression alone and thus indicated that a part of the VAP-1 pool is translocated to the endothelial cell surface (arrow). A signal is also detected on the abluminal side, indicating that the antibody gained access via inter-endothelial junctions. (**C**) Anti-VAP-1 staining followed by the secondary antibody. Negative control staining is shown in the inset. (**D**) Staining only with the secondary antibody showed no VAP-1 surface expression in the non-inflamed tissue.
