Preclinical evaluation of [⁶⁸Ga]NOTA-pentixafor for PET imaging of CXCR4 expression in vivo — a comparison to [⁶⁸Ga]pentixafor

General procedures and syntheses of the peptides are described in a recently reported protocol [21]. Determination of tracer lipophilicity [22] and serum stability [23] as well as in vitro studies were performed as recently published [23]. ⁶⁸Ga-labeling of peptides was performed as described using a fully automated system (Scintomics GmbH) [23]; briefly, the ⁶⁸Ge/⁶⁸Ga generator eluate fractions (1.25 mL) were reacted with 5 nmol of peptide. The mixture was buffered with 0.6 mL HEPES (pH = 7.4) to a final pH of 3–4 and heated to 95 °C for 5 min. After purification via one Sep-Pak C8 light cartridge, the ethanolic product fraction was diluted with PBS and used as such for the experiments.

All animal studies were conducted in accordance with the German Animal Welfare Act (Deutsches Tierschutzgesetz, approval No. 55.2-1-54-2532-71-13). For metabolite analysis, 50 MBq of [⁶⁸Ga]NOTA-pentixafor in a total volume of 200 μL of PBS was injected into the tail vein of a CB17 SCID mouse; the animal was sacrificed at 1 h p.i. and blood was collected. After sample preparation, as described in [24], the plasma samples were analyzed by reversed phase (RP)-HPLC and eluate fractions were analyzed using a γ-counter.

For PET (n = 3) and biodistribution studies (n = 5), an average of 15.2 MBq [⁶⁸Ga]NOTA-pentixafor (100 μL in PBS, 145 pmol, 171 ng) with a specific activity (A_S) of 104 GBq/μmol was injected intravenously into the tail vein of isofluorane anesthesized female Daudi lymphoma-bearing SCID mice. CXCR4 specificity of tumor accumulation was demonstrated by co-injection of 2 mg/kg AMD3100 (n = 3). After static PET imaging for 15 min (Inveon Siemens μPET scanner), the mice were sacrificed (90 min p.i.), and tissues and organs of interest were dissected, weighed, and counted for radioactivity in a γ-counter. The percentage of injected dose per gram of tissue (% ID/g) was calculated; data are shown as mean ± SD.

As already observed for [⁶⁸Ga]pentixafor (at 30 min p.i.) [5], metabolite analysis of mouse plasma at 60 min p.i. of [⁶⁸Ga]NOTA-pentixafor revealed the complete absence of radiometabolites and thus demonstrates the metabolic stability of the tracer within the observation period.

This was confirmed by small-animal PET imaging studies. Representative PET images of both tracers are shown in Fig. 2. As expected from the biodistribution data, [⁶⁸Ga]NOTA-pentixafor uptake in the Daudi xenograft was CXCR4 specific (Fig. 2b), and despite low total activity accumulation, tumors were clearly delineated 1.5 h p.i. In contrast to [⁶⁸Ga]pentixafor, however, which shows virtually no background accumulation except in kidneys [12], [⁶⁸Ga]NOTA-pentixafor also shows considerable activity accumulation in the gall bladder, intestines, and kidneys.

We recently reported the influence of different metal-chelate conjugates of pentixafor on the CXCR4 affinity [21] and found that the NOTA conjugate NOTA-pentixafor (Fig. 1) displayed the highest CXCR4 affinity among various tracers. Because NOTA offers a better suited coordination cavity for Ga³⁺ incorporation and higher thermodynamic stability and kinetic inertness compared to DOTA [18–20], [⁶⁸Ga]NOTA-pentixafor was now evaluated preclinically and compared to [⁶⁸Ga]pentixafor (Fig. 1), which is currently entering clinical studies for PET-based quantification of CXCR4 expression in vivo [5, 6, 12]. Surprisingly, despite improving CXCR4 affinity of the ligand, the exchange of DOTA by NOTA had deleterious effects on overall pharmacokinetics, both with respect to CXCR4 targeting efficiency and clearance characteristics. This is mainly attributed to the differences in chelator denticity, overall charge, and resulting changes in the complex geometry, which also affects the lipophilicity (Table 1). The increased logP of [⁶⁸Ga]NOTA-pentixafor seems to be the main reason for the enhanced background accumulation of the new compound, especially in the gallbladder and intestines. Moreover, the [Ga]NOTA-for-[Ga]DOTA exchange within the pentixafor conjugates alters the overall charge of the chelator moiety from neutral to positive which can also influence the pharmacokinetic profile. The strong dependence of the pharmacokinetics on the chelator and radiometal have also been reported for somatostatin [25, 26] or bombesin-targeting peptides [27]. In contrast, the unexpectedly low tumor accumulation of [⁶⁸Ga]NOTA-pentixafor in the Daudi xenograft model may be mainly attributed to the substantially decreased internalization efficiency of [⁶⁸Ga]NOTA-pentixafor compared to [⁶⁸Ga]pentixafor, which also seems affected by the structural changes induced by the NOTA-for-DOTA substitution. Such substantial influence of the chelator on the biodistribution has also been shown in human epidermal growth factor receptor type 2 (HER2)-targeting affibodies with DOTA, NOTA, or NODAGA-conjugates [28] as well as other GPCR ligands such as somatostatin receptor-targeting ⁶⁸Ga-labeled [Tyr³]octreotide [25].

Despite improved CXCR4 affinity, [⁶⁸Ga]NOTA-pentixafor showed severely compromised CXCR4 targeting efficiency compared to the parent compound [⁶⁸Ga]pentixafor, both in vitro and in vivo. Alongside, a substantially decreased uptake in CXCR4-positive lymphoma xenografts, [⁶⁸Ga]NOTA-pentixafor also shows enhanced accumulation in the excretory organs, leading to low tumor/background ratios and inferior imaging contrast compared to [⁶⁸Ga]pentixafor. The present data on [⁶⁸Ga]NOTA-pentixafor underline the strong dependence of the pharmacokinetics of pentixafor-based peptides on the chelator and radiometal and highlight the outstanding characteristics of [⁶⁸Ga]pentixafor for successful CXCR4 imaging.