Preclinical Development of Novel PSMA-Targeting Radioligands

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Preclinical Development of Novel PSMA-Targeting Radioligands: Modulation of Albumin-Binding Properties to Improve Prostate Cancer Therapy Christoph A. Umbricht, Martina Benesova, Roger Schibli, and Cristina Müller Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00152 • Publication Date (Web): 23 Apr 2018 Downloaded from http://pubs.acs.org on April 24, 2018

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Molecular Pharmaceutics

Preclinical Development of Novel PSMA-Targeting Radioligands: Modulation of Albumin-Binding Properties to Improve Prostate Cancer Therapy

Christoph A. Umbricht1, Martina Benešová1,2, Roger Schibli1,2, Cristina Müller1,2*

1. Center for Radiopharmaceutical Sciences ETH-PSI-USZ, Paul Scherrer Institut, 5232 Villigen-PSI, Switzerland 2. Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zurich, Switzerland

E-Mail addresses: [email protected]; [email protected]; [email protected]; [email protected]

*Correspondence to: PD Dr. Cristina Müller Center for Radiopharmaceutical Sciences ETH-PSI-USZ Paul Scherrer Institut 5232 Villigen-PSI Switzerland e-mail: [email protected] phone: +41-56-310 44 54; fax: +41-56-310 28 49

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ABSTRACT The treatment of metastatic castration-resistant prostate cancer (mCRPC) remains challenging with current treatment options. The development of more effective therapies is, therefore, urgently needed. Targeted radionuclide therapy with prostate-specific membrane antigen (PSMA)-targeting ligands has revealed promising clinical results. In an effort to optimize this concept, it was the aim of this study to design and investigate PSMA ligands comprising different types of albumin binders. PSMA-ALB-53 and PSMA-ALB-56 were designed by combining the glutamate-urea PSMA-binding entity, a DOTA chelator and an albumin binder based on the 4-(p-iodophenyl)-moiety or p-(tolyl)moiety. The compounds were labeled with

177

Lu (50 MBq/nmol) resulting in radioligands of high

radiochemical purity (≥98%). Both radioligands were stable (≥98%) over 24 h in the presence of Lascorbic acid. The uptake into PSMA-positive PC-3 PIP tumor cells in vitro was in the same range (54–58%) for both radioligands, however,

177

Lu-PSMA-ALB-53 showed a 15-fold enhanced binding

to human plasma proteins. Biodistribution studies performed in PC-3 PIP/flu tumor bearing mice revealed high tumor uptake of

177

Lu-PSMA-ALB-53 and

177

Lu-PSMA-ALB-56, respectively,

demonstrated by equal areas under the curves (AUCs) for both radioligands. The increased retention of 177Lu-PSMA-ALB-53 in the blood resulted in almost 5-fold lower tumor-to-blood AUC ratios when compared to 177Lu-PSMA-ALB-56. Kidney clearance of

177

Lu-PSMA-ALB-56 was faster and, hence,

the tumor-to-kidney AUC ratios 3-fold higher than in the case of

177

Lu-PSMA-ALB-53. Due to the

more favorable tissue distribution profile, 177Lu-PSMA-ALB-56 was selected for a preclinical therapy study in PC-3 PIP tumor-bearing mice. The tumor growth delay after application of ALB-56 and

177

177

Lu-PSMA-

Lu-PSMA-617 applied at the same activities (2 MBq or 5 MBq per mouse) revealed

better anti-tumor effects in the case of treated with

177

177

Lu-PSMA-ALB-56. As a consequence, the survival of mice

Lu-PSMA-ALB-56 was prolonged when compared to the mice which received the

same activity of 177Lu-PSMA-617. Our results demonstrated the superiority of

177

Lu-PSMA-ALB-56 over

177

Lu-PSMA-ALB-53

indicating that the p-(tolyl)-moiety was more suited as an albumin binder to optimize the tissue distribution profile.

177

Lu-PSMA-ALB-56 was more effective to treat tumors than


177

Lu-PSMA-617

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resulting in complete tumor remission in four out of six mice. This promising results warrant further investigations to assess the potential for clinical application of 177Lu-PSMA-ALB-56.

KEYWORDS: prostate cancer, PSMA ligands, radionuclide therapy, 177Lu, albumin binder



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INTRODUCTION The prostate-specific membrane antigen (PSMA) is a cell surface glycoprotein, which is physiologically expressed in normal prostate tissue and overexpressed in the majority of prostate cancer cases.1, 2 It was revealed that PSMA expression levels correlate with the stage of the disease and the risk of disease progression.3, 4 PSMA has, therefore, emerged as a promising target for nuclear imaging and targeted radionuclide therapy of metastasized castration-resistant prostate cancer (mCRPC).5-7 Over the years, a large number of PSMA-targeting radioligands have been developed for Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) imaging.5, 7-9 Among those was 68Ga-PSMA-11 which can be considered as the current “gold standard” for assessing the stage and progression of the disease as well as for monitoring the tumor response after therapeutic interventions in prostate cancer patients.10-13 PSMA-617 and PSMA I&T are ligands with a DOTA and DOTAGA chelator, respectively, which have been used for targeted radionuclide therapy of mCRPC in clinics.14-16 The ligands were labeled with

177

Lu (T1/2 = 6.65 d), a clinically-

established radionuclide which emits β¯-particles of ideal energies (Eβ͞av = 134 keV, I = 100%) for therapeutic purposes. Due to the excellent features of PSMA as a prostate cancer target and the impressive clinical results achieved with PSMA radioligands, we wanted to investigate whether the concept of PSMA-targeted radionuclide therapy could be further improved with regard to the accumulation and retention of radioligands in the tumor tissue. Previous preclinical research conducted in our group demonstrated the benefit of integrating an albumin binder into the structure of folate radioconjugates in order to improve their tissue distribution profiles.17 The research efforts resulted in radiofolates characterized with significantly increased accumulation in the tumor tissue.17, 18 The concept of using an albumin binder to improve the tissue distribution profile was adopted by other research groups and employed for the design of novel PSMA targeting ligands. Kelly et al. developed urea-based PSMA ligands with the 4-(p-iodophenyl)-moiety as an albumin binder and used this group at the same time as the carrier-moiety of [131I]iodine. 19 Choy et al. developed the first DOTA-functionalized PSMA ligand (CTT1403) with an albumin binder which was suitable for

177

Lu-labeling.20 Due to the low stability of the phosphoramidate-based


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PSMA binding entity, the preparation of

177

Lu-CTT1403 was, however, only accessible in a two-step

procedure.20 Our aim has always been the development of albumin-binding PSMA ligands equipped with a DOTAchelator to enable the labeling with a diversity of radiometals, providing in vitro and in vivo properties that would allow future clinical translation. Recently, we have published the design and evaluation of three PSMA-targeting ligands (177Lu-PSMA-ALB-02/05/07) modified with a 4-(p-iodophenyl)-based albumin binder21 and different linker units to investigate their influence on the overall tissue distribution profile.22 As compared to

177

Lu-PSMA-617, the albumin-binding PSMA radioligands

showed enhanced blood circulation and, as a consequence, significantly increased tumor accumulation.

22

The most promising candidate of this initial study,

177

Lu-PSMA-ALB-02, showed

high tumor-to-background ratios, however, the renal uptake was still relatively high. This situation was unfavorable as it may comprise a risk of renal damage when the PSMA ligand would be employed at high activities for therapeutic purposes. To address this situation, further PSMA radioligands were designed in order to find an optimum between plasma protein-binding properties and clearance of radioactivity from background tissues and organs. In this context, it was believed that modulation of the albumin-binding structure would give insight into structure/kinetics relationship of PSMA-targeting radioligands. Dumelin et al. identified a series of small molecules providing different albumin-binding affinities.21 The highest affinity was reported for 4-(p-iodophenyl)butyric acid which had the smallest dissociation constant (Kd = 3 µM).21 Other entities showed reduced albumin-binding affinity, among those the p(tolyl)butyric acid with a more than one order of magnitude higher Kd value.21 In this study, two novel PSMA radioligands comprising a 4-(p-iodophenyl)-moiety21 or a p-(tolyl)moiety21 resulting in PSMA-ALB-53 and PSMA-ALB-56, respectively, were synthesized on solidphase support as previously reported (Figure 1).22, 23 The ligands were labeled with

177

Lu for in vitro

and in vivo studies. Extensive preclinical studies were performed to determine the tissue distribution profiles of both radioligands in tumor-bearing mice. The more favorable radioligand with regard to its tissue distribution profile was applied in a site-by-site comparison to the clinically employed PSMA-617 in order to investigate the therapeutic potential in tumor-bearing mice. 5 ACS Paragon Plus Environment

177

Lu-


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Figure 1

MATERIAL AND METHODS Synthesis of PSMA Ligands. PSMA-ALB-53 and PSMA-ALB-56 were prepared using the method of solid-phase synthesis as previously reported.22The detailed synthesis of these ligands is described in the Supporting Information (Scheme S1). The compounds were purified using semipreparative RP-HPLC and characterized by analytical RP-HPLC and MALDI-MS (Supporting Information).

Radiolabeling, Stability and Distribution Coefficient. The novel PSMA ligands, PSMA-ALB53 and PSMA-ALB-56, as well as PSMA-617 (Advanced Biochemical Compounds, ABX GmbH, Radeberg, Germany) were dissolved in MilliQ water containing up to 5.5% sodium acetate (0.5 M, pH ~8) and up to 23% dimethyl sulfoxide in order to obtain 1 mM stock solutions. The radiolabeling was performed under standard labeling conditions at pH 4.5.

177

Lu (no-carrier-added

177

LuCl3 in

0.04 M HCl; Isotope Technologies Garching, ITG GmbH, Garching, Germany) was added to a mixture of sodium acetate (0.5 M, pH ~8) and HCl (0.05 M) containing the respective PSMA ligand to obtain specific activities of 5–50 MBq/nmol. The reaction mixture was incubated for 10 min at 95 °C, followed by quality control using analytical RP-HPLC (Supporting Information, Figure S1). The radioligands were used for in vitro and in vivo experiments without further purification. The PSMA ligands were labeled with

177

Lu in a volume of 120 µL at a specific activity of

50 MBq/nmol in the absence or presence of L-ascorbic acid (3 mg) to investigate the radioligand’s in vitro stability. After quality control performed using RP-HPLC (t = 0, radiochemical purity ≥98%), the labeling solutions were diluted with saline to a radioactivity concentration of 250 MBq/500 µL and incubated at room temperature. The integrity of the radioligands was determined by RP-HPLC over time (t = 1 h, 4 h and 24 h, respectively) as previously reported.18 The HPLC chromatograms were analyzed by integration of the peaks representing the radiolabeled product, the released

177

Lu as well

as degradation products of unknown structure. A quantitative assessment was performed by expressing 6 ACS Paragon Plus Environment

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the peak area of the intact product as percentage of the sum of integrated peak areas of the entire chromatogram. Determination of the n-octanol/PBS distribution coefficients (logD) was performed as previously reported (Supporting Information).22

Binding to Albumin in Human and Mouse Plasma. Experiments were performed by incubation of the 177Lu-labeled PSMA ligands (~500 kBq, 25 µL, 0.01 nmol) in different dilutions of human (Stiftung Blutspende SRK Aargau-Solothurn, Switzerland) and mouse plasma (Rockland, USA). Determination of the amount of human serum albumin (HSA) and mouse serum albumin (MSA) using a dry chemistry analyzer (DRI-CHEM 4000i, FUJIFILM, Japan) revealed values of 850 µM and 550 µM, respectively. The molar ratios of HSA-to-ligand and MSA-to-ligand were defined based on the albumin concentration. Dilutions of the human and mouse plasma containing the PSMA radioligand (HSA/MSA-to-ligand ratios in the range of 0.01–21250) were incubated for 30 min at 37 °C. Aliquots of the solutions were loaded to the ultrafiltration device (Centrifree ultrafiltration devices; 30’000 Da nominal molecular weight limit, methyl-cellulose micropartition membranes, Millipore) and centrifuged at 800 rcf for 40 min at 4 °C. The radioactivity in the filtrate as well as aliquots of the incubation solutions were measured in a γ-counter (Perkin Elmer, Wallac Wizard 1480). The albuminbound fraction was calculated based on the measurement of the filtrates, which corresponded to the free fractions, and expressed as the percentage of total added activity which was set to 100%. Unspecific binding of the radioligands to the filter membrane was not taken into consideration for this experiment as it was shown that >95% of the radioligands incubated in PBS passed the filter membrane (data not shown). The results were presented as average ± standard deviation (SD) of at least three independent experiments. The data was fit to a semi-logarithmic plot using non-linear regression (one-site, specific binding) to obtain the half maximum binding (B50) using GraphPad Prism software (version 7).



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Cell Culture and Experiments. Sublines of the androgen-independent PC-3 human prostate cancer cell line, PSMA-positive PC-3 PIP and PSMA-negative PC-3 flu cells, were kindly provided by Prof. Dr. Martin Pomper (John Hopkins Institutions, Baltimore, USA). The cells were cultured in RPMI-1640 cell culture medium supplemented with 10% fetal calf serum, L-glutamine and antibiotics. Puromycin (2 µg/mL) was added to the cell cultures to maintain PSMA expression as previously reported.24 Uptake and internalization studies of the

177

Lu-labeled PSMA ligands were performed as

previously reported (Supporting Information).22

In Vivo Studies. In vivo experiments were approved by the local veterinarian department and conducted in accordance with the Swiss law of animal protection. Mice were obtained from Charles River Laboratories (Sulzfeld, Germany) at the age of 5–6 weeks. Biodistribution and SPECT imaging studies were performed with female, athymic Balb/c nude mice subcutaneously inoculated with PSMA-positive PC-3 PIP tumor cells (6×106 cells in 100 µL Hank’s balanced salt solution (HBSS) with Ca2+/Mg2+) on the right shoulder and with PSMA-negative PC-3 flu tumor cells (5×106 cells in 100 µL HBSS with Ca2+/Mg2+) on the left shoulder. Therapy studies were performed with mice inoculated with PC-3 PIP cells (6×106 cells in HBSS with Ca2+/Mg2+) on the right shoulder.

Biodistribution Studies. Biodistribution studies were performed 12–14 days after tumor cell inoculation when the tumor xenografts reached an average mass of 123 ± 56 mg corresponding to a tumor volume of about 70–180 mm3. The PSMA ligands were labeled at a specific activity of 5 MBq/nmol and diluted in saline containing 0.05% bovine serum albumin (BSA). Tumor-bearing mice were intravenously injected with the respective radioligand (5 MBq, 1 nmol, 100 µL). The mice were sacrificed at 1 h, 4 h, 24 h, 48 h, 96 h and 192 h after injection (p.i.) and selected tissues and organs were collected, weighed and measured using a γ-counter (Perkin Elmer, Wallac Wizard 1480). Groups of 3–6 mice were used for each time point. The results were decay-corrected and listed as percentage of the injected activity per gram of tissue mass (% IA/g). Data presented as the average ± standard deviation (SD). The area under the curve (AUC) was determined for the uptake of

177

Lu-

PSMA-ALB-53 and 177Lu-PSMA-ALB-56, respectively, in PC-3 PIP tumors, kidneys and blood based 8 ACS Paragon Plus Environment

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on non-decay-corrected data obtained from the biodistribution data using GraphPad Prism software (version 7). The data sets were analyzed for significance using a one-way ANOVA with Tukey’s multiple comparison post-test using GraphPad Prism software (version 7). A p-value of 93%) at room temperature, but showed radiolytic degradation when incubated for 24 h. In the presence of L-ascorbic acid they were stable for at least 24 h (≥96%) and did not show any signs of radiolytic degradation even at high activity concentrations (250 MBq/500 µL) (Supporting Information, Figure S2).

Binding to Albumin in Mouse and Human Plasma. In different dilutions of human plasma samples, the half-maximum binding (B50) was observed at a HSA-to-ligand ratio of 51 for PSMA-ALB-53, 509 for value of

177

177

Lu-PSMA-ALB-56 and 7578 for

177

177

Lu-

Lu-PSMA-617. Relative to the B50

Lu-PSMA-ALB-56, the plasma protein-binding affinity of 177Lu-PSMA-ALB-53 was ~10-

fold increased. The B50 value of

177

Lu-PSMA-617 was ~15-fold lower than the B50 of

ALB-56 and, hence, about ~150-fold lower than the B50 value of

177

177

Lu-PSMA-

Lu-PSMA-ALB-53 (Figure 2A).

The half-maximum binding in mouse plasma samples was observed at ratios of 53 for

177

Lu-PSMA-

ALB-53 and 923 for 177Lu-PSMA-ALB-56, whereas in the case of 177Lu-PSMA-617, a ratio could not be determined as the binding remained below 6%. Relative to the B50 value of the B50 value of 177Lu-PSMA-ALB-53 was nearly 18-fold higher (Figure 2B).


177

Lu-PSMA-ALB-56,


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Figure 2

Cell Uptake and Internalization. The PC-3 PIP tumor cell uptake of 177

177

Lu-PSMA-ALB-53 and

Lu-PSMA-ALB-56 was high (48–50%) already after 2 h incubation and slightly increased up to 4 h

(55–59%). These findings were comparable to those obtained with 177Lu-PSMA-617. The internalized fraction of the new PSMA radioligands was between 15–22% after 2 h and 17–25% after 4 h. The uptake of the PSMA radioligands in PC-3 flu tumor cells was below 0.5% (Supporting Information, Figure S3).

Biodistribution Study. The tissue distribution of

177

Lu-PSMA-ALB-53 and

177

Lu-PSMA-ALB-56

was investigated in PC-3 PIP/flu tumor-bearing mice over a period of 8 days (192 h p.i.) (Figure 3, Supporting Information, Tables S2 & S3). The uptake of

177

Lu-PSMA-ALB-53 in PC-3 PIP tumors

increased within the first two days until it reached ~75% IA/g. High retention of radioactivity was observed over the following two days (75.7 ± 2.46% IA/g) with a slow wash-out over the next four days (50.4 ± 17.2% IA/g, 192 h p.i.). In the case of

177

Lu-PSMA-ALB-56, equally high tumor uptake

was observed already a few hours after injection (76.1 ± 7.67% IA/g, 4 h p.i.) with the highest tumor accumulation determined one day after injection (103.9 ± 9.06% IA/g, 24 h p.i.), however, the washout was faster resulting in ~50% of the maximum value after four days (54.5 ± 13.0% IA/g, 96 h p.i.) and ~30% of the maximum uptake after eight days (32.8 ± 9.72% IA/g, 192 h p.i.). The uptake of radioactivity in PC-3 flu tumors of mice injected with

177

Lu-PSMA-ALB-53 and

177

Lu-PSMA-ALB-

56, respectively, remained below blood activity levels throughout the entire time of investigation. High blood radioactivity levels (22–27% IA/g) were observed 1 h after injection of both radioligands, however, clearance from the blood was much faster in the case of after injection of

177

177

Lu-PSMA-ALB-56. Two days

Lu-PSMA-ALB-53, the blood activity level was still high (>10% IA/g, 48 h p.i.)

whereas only negligible radioactivity (0.05).


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Table 2. Analytical Data of PSMA-ALB-53 and PSMA-ALB-56

a

Compound

Chemical Formula

MW [g/mol]

m/za

trb [min]

Yield [%]

Chemical Purityc [%]

PSMA-ALB-53

C65H92IN11O18

1442.41

1443.57

9.5

19.2

>99.9

PSMA-ALB-56

C66H95N11O18

1330.55

1331.69

9.2

19.8

>99.2

+

Mass spectrometry of the unlabeled ligand detected as [M+H] ;

b

Retention time of unlabeled ligand on

analytical RP-HPLC. Analytical column (100×4.6 mm) utilized Chromolith RP-18e stationary phase with mobile phases consisting of 0.1% TFA in MilliQ water (A) and acetonitrile (B). A linear gradient of solvent A (90–10% over 15 min) in solvent B at a flow rate of 1 mL/min was used for analytical HPLC; c Determined by analytical RP-HPLC.



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Table 3. Area Under the Curve (AUC, in (% IA/g)·h) Based on Non-Decay-Corrected Biodistribution Data 177

Lu-PSMA-ALB-53

177

Lu-PSMA-ALB-56

PC-3 PIP Tumor

8141 ± 548

8491 ± 537

Blood

1556 ± 77

341 ± 17

Kidneys

2430 ± 121

809 ± 43

325 ± 23

73 ± 15

Tumor-to-blood

5.2

25

Tumor-to-kidney

3.4

11

Tumor-to-liver

25

116

Liver


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Table 4. Tumor Growth Inhibition (TGI) and Tumor Growth Delay Index with x-fold Increase of Tumor Size (TGDIx) of 177Lu-PSMA-ALB-56 and 177Lu-PSMA-617 First mouse Group

Treatment Group

A

1

Saline

of group

Median

euthanized

Survival

[d]

[d]

16

TGDI2

TGDI5

18

1.0 ± 0.8

1.0 ± 0.1

B

177

Lu-PSMA-617

12

19

0.8 ± 0.3

1.4 ± 0.1

C

177

Lu-PSMA-617

26

32

2.1 ± 0.3

2.5 ± 0.3

D

177

Lu-PSMA-ALB-56

28

36

1.8 ± 0.5

2.3 ± 0.6

E

177

Lu-PSMA-ALB-56

58

n.d.1

n.d.1

n.d.1

n.d. = not defined since mice were still alive at the end of the study.



Figure 1: Chemical structure of (A) PSMA-ALB-53 and (B) PSMA-ALB-56. The stronger albumin-binding 4-(piodophenyl)-moiety is highlighted in red and the weaker albumin-binding p-(tolyl)-moiety is highlighted in in blue. 673x216mm (300 x 300 DPI)

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Results of ultrafiltration assays performed with 177Lu-PSMA-ALB-53, 177Lu-PSMA-ALB-56 and 177Lu-PSMA617, respectively, to calculate the half-maximum binding (B50). (A) Binding curves of 177Lu-PSMA-ALB-53 (B50 = 51), 177Lu-PSMA-ALB-56 (B50 = 509) and 177Lu-PSMA-617 (B50 = 7578) when incubated in human plasma (average ± SD, n ≥3); (B) Binding curves of 177Lu-PSMA-ALB-53 (B50 = 53), 177Lu-PSMAALB-56 (B50 = 996) and 177Lu-PSMA-617 (B50 not defined) when incubated in mouse plasma (average ± SD, n ≥3). 126x184mm (300 x 300 DPI)



Figure 3: Biodistribution data of the radioligands obtained in Balb/c nude mice bearing PC-3 PIP and PC-3 flu tumor xenografts on the right and left shoulder, respectively. (A) Time-dependent biodistribution of 177LuPSMA-ALB-53 (5 MBq, 1 nmol). (B) Time-dependent biodistribution of 177Lu-PSMA-ALB-56 (5 MBq, 1 nmol). The values represent the average ± SD of values obtained from n = 3–6 mice. 178x105mm (300 x 300 DPI)


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Figure 4: SPECT/CT images as maximum intensity projections (MIPs) of PC-3 PIP/flu tumor-bearing mice at different time points after injection of 177Lu-PSMA-ALB-53 and 177Lu-PSMA-ALB-56. (A/B/C) MIPs of a mouse at (A) 4 h, (B) 24 h and (C) 72 h after injection of 177Lu-PSMA-ALB-53 (25 MBq, 1 nmol). (D/E/F) MIPs of a mouse at (D) 4 h, (E) 24 h and (F) 72 h after injection of 177Lu-PSMA-ALB-56 (25 MBq, 1 nmol). PSMA+ = PSMA-positive PC-3 PIP tumor; PSMA- = PSMA-negative PC-3 flu tumor; Ki = kidney; Bl = urinary bladder. 161x228mm (150 x 150 DPI)



Figure 5: Therapy study performed with 177Lu-PSMA-ALB-56 and 177Lu-PSMA-617 in PC-3 PIP tumorbearing mice. (A) Tumor growth curves relative to the tumor volume at Day 0 (set to 1) for mice that received saline (Group A), mice treated with 2 MBq 177Lu-PSMA-617 (Group B), 5 MBq 177Lu-PSMA-617 (Group C), 2 MBq 177Lu-PSMA-ALB-56 (Group D) and 5 MBq 177Lu-PSMA-ALB-56 (Group E). Data are shown until the first mouse of the respective group reached an endpoint. (B) Kaplan-Meier plot of Groups A– E. (C) Relative body weight of Groups A–E. 146x279mm (300 x 300 DPI)


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TOC 121x39mm (150 x 150 DPI)


Preclinical Development of Novel PSMA-Targeting Radioligands

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