Design and Preclinical Evaluation of an Albumin-Binding PSMA

Article Cite This: Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Design and Preclinical Evaluation of an Albumin-Binding PSMA Ligand for 64Cu-Based PET Imaging Christoph A. Umbricht,†,∥ Martina Benešova,́ †,‡,∥ Roger Hasler,† Roger Schibli,†,‡ Nicholas P. van der Meulen,†,§ and Cristina Müller*,†,‡ Center for Radiopharmaceutical Sciences ETH-PSI-USZ and §Laboratory of Radiochemistry, Paul Scherrer Institute, 5232 Villigen-PSI, Switzerland ‡ Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zurich, Switzerland Downloaded via KAOHSIUNG MEDICAL UNIV on October 31, 2018 at 05:40:51 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

†

S Supporting Information *

ABSTRACT: Recently, we developed an albumin-binding radioligand (177Lu−PSMA−ALB-56), which showed higher PSMA-specific tumor uptake in mice than the previously developed 177Lu−PSMA-617 under the same experimental conditions. Such a radioligand may be of interest also for PET imaging, possibly enabling better visualization of even small metastases at late time-points after injection. The aim of this study was, therefore, to modify PSMA−ALB-56 by exchanging the DOTA chelator with a NODAGA chelator for stable coordination of 64 Cu (T1/2 = 12.7 h; Eβ+av = 278 keV). The resulting NODAGAfunctionalized PSMA−ALB-89 ligand, and the previously establish DOTA-functionalized PSMA−ALB-56 ligand were labeled with 64Cu and evaluated in vitro and in vivo. Both radioligands showed plasma protein-binding properties in vitro and PSMA-specific uptake in PC-3 PIP cells. Biodistribution studies, performed in tumor-bearing mice, revealed high accumulation of 64Cu−PSMA−ALB-89 in PSMA-positive PC-3 PIP tumor xenografts (25.9 ± 3.41% IA/g at 1 h p.i.), which was further increased at later time-points (65.1 ± 7.82% IA/g at 4 h p.i. and 97.1 ± 7.01% IA/g at 24 h p.i.). High uptake of 64Cu−PSMA−ALB-89 was also seen in the kidneys, however, 64Cu−PSMA−ALB-89 was efficiently excreted over time. Mice injected with 64Cu−PSMA−ALB-56 showed increased accumulation of radioactivity in the liver (25.3 ± 4.20% IA/g) when compared to the liver uptake of 64Cu−PSMA− ALB-89 (4.88 ± 0.21% IA/g, at 4 h p.i.). This was most probably due to in vivo instability of the 64Cu−DOTA complex, which was also the reason for lower tumor uptake (49.7 ± 16.1% IA/g at 4 h p.i. and 28.3 ± 3.59% IA/g at 24 h p.i.). PET/CT imaging studies confirmed these findings and enabled excellent visualization of the PSMA-positive tumor xenografts in vivo after injection of 64Cu−PSMA−ALB-89. These data indicate that 64Cu−PSMA−ALB-89 is favorable over 64Cu−PSMA−ALB-56 with regard to the in vivo stability and tissue distribution profile. Moreover, 64Cu−PSMA−ALB-89 outperformed previously developed 64Cu-labeled PSMA ligands. Further optimization of long-circulating PSMA-targeting PET radioligands will be necessary before translating this concept to the clinics. KEYWORDS: prostate cancer, PSMA, PET imaging, copper-64, 64Cu, albumin binder

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INTRODUCTION

nontargeted organs increases with time, small metastases may be better detectable at late time-points after injection.13,14 The short half-life of 68Ga (T1/2 = 68 min) limits, however, the option of performing PET imaging at late time-points after injection of this radioligand.14 Rapid blood clearance of small-molecule-based PSMA radioligands15 is limiting for the accumulation of radioactivity in the tumor tissue, which may present another shortcoming for the detection of small metastases. A possibility to improve this situation could be the upregulation of PSMA in the tumor

The prostate-specific membrane antigen (PSMA) has been found overexpressed in over 90% of metastatic castrationresistant prostate cancer (mCRPC) cases.1,2 Moreover, it was reported that PSMA expression levels correlate with differentiation and androgen independence of the disease.3−5 In recent years, a large number of low-molecular-weight imaging agents and therapeutics were developed for PSMA targeting, among those also candidates which were already applied in patients.6−10 A major breakthrough in this field was the clinical translation of 68Ga−PSMA-11, which is currently employed in many hospitals worldwide for diagnostic imaging and pre/posttherapeutic staging of the disease.11,12 As the contrast between accumulated radioactivity in the tumor tissue and uptake in © XXXX American Chemical Society

Received: July 8, 2018 Revised: October 8, 2018 Accepted: October 15, 2018

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DOI: 10.1021/acs.molpharmaceut.8b00712 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Article

Molecular Pharmaceutics

Figure 1. Chemical structures of PSMA ligands. (A) PSMA−ALB-89, the herein described PSMA ligand with a NODAGA chelator (red); (B) PSMA−ALB-56,23 a previously developed PSMA ligand with a DOTA chelator (blue).

Scheme 1. Conjugation of NODAGA Chelator for the Synthesis of PSMA−ALB-89a

a

(a) NODAGA−tris(t-Bu) ester, HBTU, DIPEA, DMF; (b) TFA, TIPS, Milli-Q water, 95:2.5:2.5.

radionuclides to obtain high imaging contrast at later timepoints after injection of the radioligand. In this regard, 64Cu appeared most appropriate, as it decays by the emission of β+particles of low energy (Eβ+mean = 278 keV, I = 17.6%), which is particularly suitable for high-resolution PET. The relatively long half-life (T1/2 = 12.7 h) of 64Cu makes it useful for imaging purposes, even the day after application, which is not possible with short-lived PET nuclides, such as 68Ga (T1/2 = 68 min) or 18F (T1/2 = 110 min). Another PET radiometal with an even longer half-life is 89Zr (T1/2 = 78.4 h, Eβ+av= 396 keV, I = 22.7%) which is commonly coordinated using desferoxamine (DFO) as a chelator.25 89Zr proved particularly promising in combination with antibodies that have shown great potential for cancer imaging using immuno-PET.26,27 The DOTA chelator has proven useful for the coordination of frequently employed radionuclides including 111In, 68Ga, 90 Y, and 177Lu;28 hence, a large number of targeting agents have been functionalized with this chelator.29 Several of these ligands, including PSMA-617, were labeled with 64Cu and used in preclinical and clinical studies.30,31 An increasing body of evidence indicates, however, low in vivo stability of 64Cu− DOTA complexes, resulting in unfavorable image contrast.28,32 On the basis of preclinical data, it was known that released copper accumulates primarily in the liver, which was meanwhile confirmed clinically in a study with healthy volunteers who received 64CuCl2.33 This was also a major concern when using 64Cu-labeled PSMA-617, since high radioactivity uptake was observed in the liver of mice as well as patients, which was not the case when using PSMA-617 labeled with 177Lu.34−36 In contrast, coordination of 64Cu by 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODA-

tissue, as it has been proposed recently through use of specific pharmaceuticals including hormone-active drugs.16−18 In view of a therapeutic application, albumin-binding PSMA ligands were developed to enhance the blood circulation time and to increase tumor accumulation of radioactivity.19,20 Our group was the first to design albumin-binding PSMA ligands based on the glutamate−urea-binding entity and a 1,4,7,10tetraazacyclododecane−1,4,7,10-tetraacetic acid (DOTA) chelator for labeling with 177Lu (T1/2 = 6.65 days, Eβ¯av = 134 keV, Eγ = 113, 208 keV).21 Subsequently, Kelly et al. reported on the development of additional PSMA ligands equipped with an albumin-binding entity.22 These ligands were labeled with 177 Lu and 66Ga for preclinical evaluation. A significantly increased tumor uptake was observed after injection of 177 Lu−RPS-063 in LNCaP tumor xenografts of mice as compared to the uptake of 177Lu−PSMA-617.22 Among the five published PSMA ligands developed in our group, PSMA− ALB-56 was identified as the lead compound.23 It consists of a DOTA chelator and a weak albumin binder based on a p-tolyl moiety, which was previously described by Dumelin et al.23,24 Application of 177Lu−PSMA−ALB-56 in a therapeutic setting showed better effects on PSMA-positive tumor xenografts in mice than were observed when using 177Lu−PSMA-617.23 177 Lu−PSMA−ALB-56 showed excellent tumor-to-background contrast on SPECT/CT images at later time-points when the majority of blood activity was already cleared.23 On the basis of these findings of two independent groups, the application of long-circulating PSMA-targeting radioligands may also be a valid concept for PET imaging, possibly with the potential to identify more lesions in patients with mCRPC. This would require, however, the application of longer-lived B


Article


over 4 h after preparation. All detected peaks on the HPLC chromatogram were integrated and collectively set to 100%. The amount of intact radioligand was quantified by integration of the product peak in relation to the sum of all peaks of radioactive degradation products of unknown structure and traces of released 64Cu. Determination of n-Octanol/PBS Distribution Coefficients (LogD Values). The n-octanol/PBS (pH 7.4) distribution coefficients (logD values) of the 64Cu-labeled radioligands were determined by a shake-flask method using liquid−liquid extraction followed by phase separation as previously reported (Supporting Information).41 Three experiments were performed with five replicates for each radioligand. The data were analyzed for statistical significance using an unpaired t test (GraphPad Prism software, version 7). A pvalue 98%) and similar retention times (∼11 min; Figure S1). 64Cu−PSMA−ALB-89 and 64Cu− PSMA−ALB-56 were stable (>92%) over a period of at least 4 h. The n-octanol/PBS distribution coefficient (logD value) of 64 Cu−PSMA−ALB-89 (−2.3 ± 0.7) was slightly, but not significantly (p > 0.05), higher than the logD value of 64Cu− PSMA−ALB-56 (−3.1 ± 0.1). Albumin-Binding Properties. 64Cu−PSMA−ALB-89 and 64 Cu−PSMA−ALB-56 showed high binding to plasma proteins when incubated in human plasma. Relative to the binding affinity of 64Cu−PSMA−ALB-56 (set to 1.0), an increased binding affinity was determined for 64Cu−PSMA− ALB-89 (1.7; Figure S2).

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RESULTS Synthesis of the PSMA Ligands. PSMA−ALB-89 was synthesized using a solid-phase support in analogy to the synthesis of PSMA−ALB-56 (Figure 1).23 Instead of conjugating a DOTA chelator, a NODAGA chelator was D


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Molecular Pharmaceutics Cell Uptake and Internalization. The uptake of 64Cu− PSMA−ALB-89 in PC-3 PIP cells was ∼46%, and the internalized fraction was ∼14% after an incubation period of 2 h at 37 °C. The cell uptake increased slightly after 4 h of incubation time (∼52%), while the internalized fraction remained unchanged (∼14%). Similar values were determined for 64Cu−PSMA−ALB-56 (Figure 2A). The uptake in PC-3 flu cells was below 0.5% for both radioligands, indicating that the uptake in the PC-3 PIP cell was PSMA-specific (Figure 2B). Biodistribution Study. The tissue distribution profile of 64 Cu−PSMA−ALB-89 was assessed over a period of 24 h in tumor-bearing mice and compared to the data obtained with 64 Cu−PSMA−ALB-56 (Figure 3; Tables S2 and S3). Accumulation of 64Cu−PSMA−ALB-89 in PC-3 PIP tumors was high and comparable to the uptake of 64Cu−PSMA−ALB56 at 1 h p.i. (25.9 ± 3.41 vs 24.3 ± 2.56% IA/g; p > 0.05). At later time-points, the tumor uptake of 64Cu−PSMA−ALB-89 was, however, significantly increased in comparison to the uptake of 64Cu−PSMA−ALB-56 (65.1 ± 7.82 vs 49.7 ± 16.1% IA/g, 4 h p.i.; p < 0.05 and 97.1 ± 7.01 vs 28.3 ± 3.59% IA/g, 24 h p.i.; p < 0.05). The radioactivity concentration in PC-3 flu tumors was generally below blood levels for both radioligands with the only exception being the 24 h p.i. time-point of 64Cu− PSMA−ALB-56 (Figure 3). At 1 h p.i., the radioactivity detected in the blood pool of mice injected with 64Cu− PSMA−ALB-89 was significantly higher than in mice injected with 64Cu−PSMA−ALB-56 (28.4 ± 4.04 vs 18.8 ± 1.26% IA/ g; p < 0.05); however, effective blood clearance over time was observed for both radioligands (Figure 3). At all investigated time-points, the kidney uptake of 64Cu−PSMA−ALB-89 was significantly higher than for 64Cu−PSMA−ALB-56 (65.4 ± 7.39 vs 40.2 ± 4.92% IA/g, at 1 h p.i; p < 0.05; 92.3 ± 5.17 vs 20.3 ± 4.18% IA/g at 4 h p.i; p < 0.05; and 36.7 ± 5.20 vs 8.07 ± 0.67% IA/g at 24 h p.i.; p < 0.05). On the other hand, liver uptake was significantly higher after injection of 64Cu−PSMA− ALB-56 (p < 0.05) over the whole period of investigation. The maximum liver uptake of 64Cu−PSMA−ALB-56, observed at 4 h p.i (25.3 ± 4.20% IA/g), was far above blood activity levels. In contrast, the liver uptake pattern of 64Cu−PSMA−ALB-89 revealed radioactivity levels in the range of blood pool activity or below (Figure 3). The tumor-to-muscle ratios were high already shortly after application of 64Cu−PSMA−ALB-89 and 64Cu−PSMA−ALB56 (9.04 ± 1.13 and 12.1 ± 2.5, respectively, at 1 h p.i.). These ratios were more than 20-fold increased at 24 h after injection of 64Cu−PSMA−ALB-89 but only ∼2.5-fold increased in the case of 64Cu−PSMA−ALB-56 (Table 1). The tumor-to-blood ratios of 64Cu−PSMA−ALB-89 at 1 and 4 h p.i. were lower than the ratios observed after injection of 64Cu−PSMA−ALB56; however, at 24 h p.i. the tumor-to-blood ratio of 64Cu− PSMA−ALB-89 was more than 30-fold increased relative to the study start compared to an only 15-fold increased ratio for 64 Cu−PSMA−ALB-56 (Table 1). Tumor-to-kidney ratios of both radioligands increased with time; however, the values were slightly lower after injection of 64Cu−PSMA−ALB-89 at all investigated time-points (Table 1). The tumor-to-liver ratios of 64Cu−PSMA−ALB-89 increased over time, which was in contrast to the ratios observed after injection of 64Cu− PSMA−ALB-56 that remained 1 and increased further with time. Background activity in organs and tissues stemming from the radioactivity in the blood was visible on the image taken at 1 h p.i. but disappeared at later time-points. The uptake of 64Cu−PSMA−ALB-56 in the tumor tissue was lower than in the case of 64Cu−PSMA−ALB-89, and almost no retention of 64Cu−PSMA−ALB-56 was detected in the kidneys. In line with biodistribution data, liver accumulation of 64Cu−PSMA−ALB-56 was visible on PET/CT images, predominantly at early time-points after injection.

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DISCUSSION In this work, we developed a long-circulating PSMA ligand for labeling with 64Cu to enable PET at late time-points after radioligand application. The yield of the synthesized PSMA− ALB-89 was moderate, but it may be further optimized by increasing the equivalencies of respective building blocks and prolonging the reaction times as previously stated.21,23 Radiolabeling with 64Cu was achieved reproducibly at specific activities up to 50 MBq/nmol, indicating the good chemical purity of the synthesized ligand as well as a favorable radiochemical purity of the 64Cu produced at the research cyclotron (Injector 2 facility) at the Paul Scherrer Institute. The production of 64Cu would be feasible on medical cyclotrons (11−18 MeV); however, it will require the installation of a solid target station, which is currently not available at most standard medical cyclotrons. It is likely that 64 Cu would not be made available on a daily basis at individual PET centers in the near future. As a result of its relatively long half-life of 12.7 h, centralized production of 64Cu radiopharmaceuticals and shipment to satellite PET centers without a radiopharmacy could be a practicable solution. The in vitro stabilities of 64Cu−PSMA−ALB-89 and 64Cu− PSMA−ALB-56 were in agreement with previously reported studies demonstrating high in vitro stability of 64Cu− NODAGA and 64Cu−DOTA complexes.37,39 The specific binding to PSMA was confirmed for 64Cu−PSMA−ALB-89 and 64Cu−PSMA−ALB-56 in vitro and in vivo as demonE


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Figure 4. PET/CT images shown as maximum intensity projections. (A−D) PET/CT images of a mouse 1, 4, 16, and 24 h after injection of 64Cu− PSMA−ALB-89. (E−H) PET/CT images of a mouse 1, 4, 16, and 24 h after injection of 64Cu−PSMA−ALB-56. The scale has been adjusted by cutting 2% of the background to make the tumors, kidneys, and liver better visible. (PSMA+ = PC-3 PIP tumor xenograft; PSMA− = PC-3 flu tumor xenograft; Ki = kidney; Li = liver; Bl = urinary bladder).

The high retention of 64Cu−PSMA−ALB-89 in the kidneys was unexpected and in clear contrast to the result previously obtained with 177Lu−PSMA−ALB-56.23 On the other hand, it was found that the tumor uptake of 64Cu−PSMA−ALB-89 was in the same range as 177Lu−PSMA−ALB-56.23 These findings suggest that the chelator−metal complex (Cu−NODAGA vs Lu−DOTA) plays an important role for the kidney clearance of the PSMA-targeting radioligands and should be taken into consideration in future when designing novel radioligands. As demonstrated in this particular case, the coordination chemistry may strongly vary among different radiometals and depend on the employed chelator. The Cu(II) cation has a ionic radius of 73 pm, which is smaller than the ionic radius of the Lu(III) cation (86 pm).45 In addition, Lu−DOTA results in an octacoordinated complex, where all carboxyl groups of the chelator are involved in the coordination, while in the hexacoordinated complex of Cu−NODAGA, one carboxylic group remains free. This situation may be the reason for the different in vivo behaviors of the radioligands. A similar observation was made by Banerjee et al., who investigated PSMA ligands with NOTA and DOTA chelators using 68Ga for PET imaging. Whereas the tumor uptake was in the same range for both radioligands, retention of radioactivity in the kidneys was ∼4-fold and ∼3-fold higher at 1 and 2 h p.i.,

strated by high uptake in PSMA-positive (PC-3 PIP) cells but negligible uptake in PSMA-negative (PC-3 flu) tumor cells. The tissue distribution profile of 64Cu−PSMA−ALB-89 in the PC3 PIP/flu xenograft mouse model differed considerably from the biodistribution of 64Cu−PSMA−ALB-56. The significantly higher tumor uptake of 64Cu−PSMA−ALB-89 compared to 64Cu−PSMA−ALB-56 at 4 and 24 h p.i. may be explained by the increased retention of 64Cu−PSMA−ALB-89 in the blood pool. The high liver uptake after application of 64 Cu−PSMA−ALB-56 may be a consequence of 64Cu− PSMA−ALB-56’s in vivo instability. It is generally known that 64Cu−DOTA complexes exhibit low in vivo stability and that trans-chelation of released copper to endogenous proteins may become evident by increased background activity.43 Our findings were in line with previously reported results obtained with 64Cu−PSMA-617, where copper was also coordinated by a DOTA chelator.34,36 PET/CT images obtained with both radioligands confirmed the favorable tissue distribution profile of 64Cu−PSMA−ALB-89 over 64Cu−PSMA−ALB-56. High tumor uptake and low activity accumulation in the liver are essential, giving the fact that prostate cancer may result in liver metastases,44 which could be masked by unspecific accumulation of radioactivity in the liver. F


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Molecular Pharmaceutics respectively, when using the radioligand with a NOTA chelator.46 Another study, performed in the same research group, reported on PSMA ligands with various chelators for coordination of 64Cu.47 It was found that the selected chelator had a profound effect on the radioligands’ pharmacokinetics, demonstrated by diverging values for renal uptake ranging from ∼24 to ∼166% IA/g at 2 h after injection.47 With regard to the tumor uptake, 64Cu−PSMA−ALB-89 outperformed other 64Cu-based PSMA ligands reported in the literature.35,47 Moreover, the maximum tumor uptake of 64Cu− PSMA−ALB-89 was observed at 24 h p.i.; hence, it is possible that the uptake would further increase at later time-points. Throughout the investigated period, the tumor-to-muscle ratio of 64Cu−PSMA−ALB-89 increased by a factor of 20, indicating improved contrast at later time-points after injection. The benefit of late imaging is unclear at this stage, but it may be of value to unambiguously identify unclear findings. On the basis of the biodistribution data and their comparison with currently employed PSMA-targeting radioligands (e.g., 68Ga− PSMA-11), it is questionable whether 64Cu−PSMA−ALB-89 would enable the detection of additional metastases. In addition, the time between radioligand application and late time image acquisition may pose a logistic challenge. Patient management in a clinic should be time-efficient with short intervals between injection and nuclear imaging procedures. Longer waiting times for patients may be justified, however, if there is a benefit of late-time imaging. Clinical data of albuminbinding PSMA radioligands are, however, not available yet, and hence, a potential added value of long-circulating radioimaging agents remains to be demonstrated. In summary, we have developed the currently most promising PSMA ligand to be used in combination with 64 Cu for PET imaging. The high kidney uptake of 64Cu− PSMA−ALB-89 would prevent a therapeutic application with 67 Cu (T1/2 = 61.8 h, Eβ¯av = 141 keV), which could have been employed as a therapeutic match.48 In this regard, it will be necessary to design radioligands with more favorable pharmacokinetics to enable radiotheragnostic application of 64 Cu/67Cu-based PSMA-targeting ligands.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; Phone: +41-56-310 44 54; Fax: +41-56-310 28 49 (C.M.) ORCID

Cristina Müller: 0000-0001-9357-9688 Author Contributions ∥

C.A.U. and M.B. have equally contributed to this manuscript.

Notes

The authors declare the following competing financial interest(s): Patent applications on PSMA ligands with albumin-binding entities have been filed by Isotope Technologies Munich (ITM) AG.

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ACKNOWLEDGMENTS The authors thank Walter Hirzel, Alexander Sommerhalder, Susan Cohrs, and Alexandra Christen for technical assistance of the experiments at PSI.

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REFERENCES

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CONCLUSION Cu−PSMA−ALB-89 showed increased in vivo stability as compared to 64Cu−PSMA−ALB-56 and outperformed previously developed PSMA ligands for 64Cu labeling. The increased tumor accumulation of this radioligand observed on late PET images after injection could possibly improve the visualization of PSMA-positive prostate cancer lesions. Clearly, the design of this radioligand needs to be revised before translating the “albumin binder” concept to clinics. Whether or not it would provide an advantage over currently employed PET agents for PSMA targeting in specific situations of mCRPC patients will be a topic of future investigations. 64

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methods, cell uptake and internalization, biodistribution data, additional PET data and biodistribution results (PDF)

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.8b00712. Chemicals and organic synthesis, HPLC purification and analysis, characterization of the ligands, radiolabeling and quality control of PSMA radioligands, logD values, determination of albumin-binding properties, cell culture G


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Design and Preclinical Evaluation of an Albumin-Binding PSMA

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