Cationic eluate pretreatment for automated synthesis of [68Ga]CPCR4.2

https://doi.org/10.1016/j.nucmedbio.2013.09.002Get rights and content

Abstract

Fostered by the clinical success of sst-ligands, the development and evaluation of 68Ga-labeled peptides have become a very active field in radiopharmaceutical chemistry. Consequently, various new peptide tracers have been developed, e.g. [68Ga]CPCR4.2 for in vivo imaging of solid and haematological tumors or [68Ga]TRAP(RGD)3 for imaging of αvβ3 integrin expression. As a consequence of different matrices (TiO2, SnO2, polymers) exploited in commercial 68Ge/68Ga-generators, HCl of different concentrations (0.05…1.0 M) is used to obtain 68Ga as starting material for automated syntheses. We have developed a purification method which reduces the eluate volume and adjusts the HCl concentration. The method may potentially allow standardization of the eluate composition of different commercial generators prior to labeling. Recently, a cationic purification process has been reported which allows the pre-fixation of 68Ga on a Varian SCX cation exchange cartridge and subsequent elution of 68Ga with acidified NaCl solutions. As part of the development of ready-to-use cassettes for the automated production of 68Ga-CPCR4.2 using a SCINTOMICS GRP module and an iThemba Labs generator that is eluted with 0.6…1.0 M HCl, we tested and compared the 68Ga-trapping efficiency of various commercial available cation exchange cartridges, the efficiency of subsequent 68Ga-elution from these cartridges by means of various protocols and the influence of these variations on the labeling efficiency of [68Ga]CPCR4.2, [68Ga]TRAP(RGD)3 and [68Ga]DOTATATE/[68Ga]DOTANOC. Finally, we transferred the optimized method to the automated, cassette based synthesis of [68Ga]CPCR4.2 and the aforementioned peptides. From seven tested cation exchange cartridges, Chromafix PS-H+ gave the best extraction results (> 95%). Moreover, we observed that acidified solutions of 5 M NaCl or 2.5 M CaCl2 can be used for efficient cartridge elution. Using a disposable cGMP-compliant cassette system, we obtained [68Ga]CPCR4.2 in 80% decay-corrected yield and > 99% purity. These data were confirmed by the production of [68Ga]DOTATATE, [68Ga]DOTANOC and [68Ga]TRAP(RGD)3 on the otherwise identical cassette system.

Introduction

During the last years, a variety of 68Ga-labeled peptides have been introduced as PET tracers for imaging of somatostatin expressing tumors, i.e. neuroendocrine tumors [1], [2], [3]. 68Ga (t1/2= 68 min, Eβ+max=1.89 MeV) is obtained from commercially available 68Ge/68Ga radioisotope generators. Most commonly, 68Ge is adsorbed onto column matrixes containing TiO2, SnO2 or organic polymers. From these generators, 68Ga3 + can be obtained by elution with HCl in concentrations ranging from 0.05 to 1 M. Consequently, the eluates from currently available generator systems can differ with regard to 68Ga-elution efficiency, eluate volume, HCl concentration, metal impurities and 68Ge-breakthrough. The large eluate volume and the rather low pH (0–1.5) of the eluate solution prevent the direct use for labelling reactions, and the labelling yields might not be optimal due to the presence of metallic impurities, for example Zn(II), the stable daughter of 68Ga. Therefore, cationic purification and anionic purification procedures already described in the 1960's [4], [5] were adapted for the pre-processing of a 68Ge/68Ga-generator eluate prior to peptide labeling. The cationic purification method developed by Zhernosekov et al. is based on a small cation exchange cartridge that extracts 68Ga3 + and 68Ge4 + from a 0.1 M HCl solution [6]. After a washing step with 0.15 M HCl/acetone (elution of remaining Ge, Ti, Fe, and Zn), 68Ga is eluted with a mixture of 0.05 M HCl/98% acetone [6]. However, the drawback of this methodology is the use of acetone, since acetone prevents the use of disposable cassettes not stable against this solvent. Furthermore, acetone has to be removed before injection into the patient and the residual acetone concentration in the final product needs to be quantified as part of the quality control. The methodology developed by Meyer et al. makes use of the fact that at concentrations of > 5.5 M HCl, negatively charged [68GaCl4] species can be adsorbed on anion exchange resins [7]. After purging the resins with gas, e.g. nitrogen, 68Ga3 + can be obtained by elution of the anion exchange resins with water. In contrast to the aforementioned methods [6], [7], the fractionated procedure is based on the collection of a fraction of only ~ 1–2 mL of the entire eluate volume, corresponding to 70%–80% of the maximum generator activity [8]. Obviously, using the fractionation, the maximum decay-corrected yields cannot exceed the fractionation efficiency of 70%–80%.

More recently, an alternative cationic purification method has been described by Müller et al. [9]. In this study, 68Ga, adsorbed on a SCX cation exchange column, was eluted with acidified (50 μl HCl) 5 M NaCl. However, this method has been shown to work only in combination with generators that use 0.05–0.1 M HCl for elution.

The majority of 68Ga-labeled peptides are mainly used for imaging of peptide hormone, i. e. somatostatin receptor expressing tumors. In contrast, the chemokine receptor CXCR4 plays an important role in tumor development, progression and metastasis. Additionally it is a receptor involved in vasculogenesis [10], tissue remodelling and the cell entry of the human immunodeficiency virus (HIV) [11]. Due to its excellent affinity and pharmacokinetics, [68Ga]CPCR4.2, a cyclic pentapetide ligand with high affinity for the chemokine receptor CXCR4 is the most promising 68Ga-labeled CXCR4 PET ligand currently available (Fig. 1) [12], [13], [14], [15], [16]. To support first clinical [68Ga]CPCR4.2 PET-studies, we aimed to develop ready-to-use cassettes for the automated and fully GMP-compliant production of [68Ga]CPCR4.2 using a SCINTOMICS GRP module and an iThemba Labs generator [17] that is based on a SnO2 matrix and eluted with 0.6 to 1.0 M HCl. In that context we focussed on a suitable method to reduce the eluate volume, to adjust the HCl concentration and thus to allow a standardization of the quality and composition of the 68Ge/68Ga-generator eluates, i.e. to make the labeling conditions independent from the generator used. Consequently, all commercially available generators, eluted according to the supplier's recommendations, should be connectable to the described system and should result in the same standardized 68Ga-solution for labeling.

Due to the appealing feature of an acetone-free elution, we aimed to use the cationic purification method developed by Müller D et al. [9] for standardization. Moreover, handling with concentrated hydrochloric acid, which is used during anionic purification [7], can be omitted, which minimizes potential corrosion effects caused by volatile HCl on stainless steel hot cells, as often observed over time. Furthermore, when compared with the fractionation method, the entire generator eluate can be delivered to the cation exchange cartridge, thus ensuring better overall production yields for the cationic procedure.

Section snippets

Peptides

CPCR4.2 and TRAP(RGD)3 are SCINTOMICS's peptides prepared in custom syntheses by ABX. DOTATATE and DOTANOC have been prepared by ABX GmbH, Radeberg. All peptides were obtained in > 98% purity. The plastic reactor vials were filled with aqueous solutions containing 20 μg of CPCR4.2 trifluoroacetate (16 nmol) or 20 μg of DOTATATE/DOTANOC trifluoroacetate (14 nmol) or 1 μg of TRAP(RGD)3 (0.33 nmol). After freeze-drying, the sealed plastic vials containing the lyophilisates were stored at –20°C.

68Ge/68Ga radionuclide generator

A

Results and discussion

The trapping efficiency was determined for the adsorption of 68Ga3 + from 10 mL of the generator eluate (1.0 M HCl) using 7 different SPE cation exchange cartridges (Table 1, Table 2). Only cation exchange cartridges with an adsorption efficieny of > 50% were selected for subsequent elution experiments. Both, preconditioned and non-conditioned Varian Bond Elut SCX cartridges (100 mg) showed only a low adsorption efficiency of 7% (Table 1, entry 1). This cartridge type was used for the adsorption

Conclusion

A disposable cassette system for GRP-modules with a PS-H+ SCX-cartridge based 68Ga-prepurification method has been established and used for the 68Ga-labeling of peptides. The established methodology allows for the fully automated synthesis of [68Ga]CPCR4.2 in 79% decay-corrected yield and 99% purity. As further demonstrated by the labeling of TRAP(RGD)3 and DOTATATE/DOTANOC, this cassettes system can also be applied to the synthesis of a broad range of other peptides without any specific

Acknowledgment

We would like to thank Dr. Albert Brennauer (ABX) for the synthesis of the peptides and Dr. Johannes Notni (TUM) for his valuable comments on the synthesis of TRAP(RGD)3.

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