Next Generation of SiFAlin-Based TATE Derivatives for PET Imaging

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Next generation of SiFAlin-based TATE derivatives for PET imaging of SSTR-positive tumors: Influence of molecular design on in vitro SSTR binding and in vivo pharmacokinetics Shanna Litau, Sabrina Niedermoser, Nils Vogler, Mareike Roscher, Ralf Schirrmacher, Gert Fricker, Björn Wängler, and Carmen Wängler Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.5b00510 • Publication Date (Web): 30 Sep 2015 Downloaded from http://pubs.acs.org on October 2, 2015

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Bioconjugate Chemistry

Next generation of SiFAlin-based TATE derivatives for PET imaging of SSTR-positive tumors: Influence of molecular design on in vitro SSTR binding and in vivo pharmacokinetics S. Litau,†,‡ S. Niedermoser,‡ N. Vogler,§ M. Roscher,‡ R. Schirrmacher,ǁ G. Fricker,┴ B. Wängler,*‡ C. Wängler*† †

‡

§

ǁ

┴

Biomedical Chemistry, Department of Clinical Radiology and Nuclear Medicine, Medical Faculty Mannheim of Heidelberg University, Mannheim, Germany Molecular Imaging and Radiochemistry, Department of Clinical Radiology and Nuclear Medicine, Medical Faculty Mannheim of Heidelberg University, Mannheim, Germany Nuclear Medicine, Department of Clinical Radiology and Nuclear Medicine, Medical Faculty Mannheim of Heidelberg University, Mannheim, Germany Department of Oncology, Div. Oncological Imaging, University of Alberta, Edmonton, Canada Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, Heidelberg, Germany

ABSTRACT The Silicon-Fluoride-Acceptor (SiFA)-18F-labeling strategy has been shown before to enable the straightforward and efficient 18F-labeling of complex biologically active substances such as proteins and peptides. Especially in case of peptides, the radiolabeling proceeds kit-like in short reaction times and without need of complex product workup. SiFA-derivatized, 18Flabeled Tyr3-octreotate (TATE) derivatives As shown in previous studies, the SiliconFluoride-Acceptor (SiFA)-18F-labeling strategy allows for a fast and efficient kit-like synthesis of 18F-labeled Tyr3-octreotate (TATE) derivatives. These demonstrated before , apart from strong somatostatin receptor (SSTR) binding, favorable in vivo pharmacokinetics and as well as enabled an excellent tumor visualization by PET imaging. In this study, we intended The aim of this study was to determine the influence of the underlying molecular design and used molecular scaffolds of SiFAlin-TATE derivatives on SSTR binding as well as on the in vivo pharmacokinetics of the resulting 18F-labeled peptides. For this purpose, new SiFAlin-(Asp)n-PEG1-TATE analogs (where n = 1 – 4) were synthesized, efficiently radiolabeled with 18F in a kit-like manner and obtained in radiochemical yields of 70 – 80%, radiochemical purities of ≥97% and non-optimized specific activities of 20.1 – 45.2 GBq/µmol within 20-25 minutes starting from 0.7 – 1.5 GBq of 18F. In the following, the radiotracer’s lipophilicities and stabilities in human serum were determined in vitro. Furthermore, the SSTR-specific binding affinities were evaluated by a competitive displacement assay on SSTR-positive AR42J cells. The obtained in vitro results support the 1

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assumption that the introduced aspartic acids are able to considerably increase the radiotracer’s hydrophilicity and that their number does not affect the SSTR binding potential of the TATE derivatives. The most promising tracer 18F-SiFAlin-Asp3-PEG1-TATE [18F]6 (LogD = -1.23 ± 0.03, IC50 = 20.7 ± 2.5 nM) was further evaluated in vivo in AR42J tumorbearing nude mice via PET/CT imaging against the clinical gold standard 68Ga-DOTATATE as well as an the earlier developed SiFAlin-TATE derivative [18F]3. The results of these evaluations showed that [18F]6 − although showing very similar chemical and in vitro properties to [18F]3 − exhibits a slowed renal clearance compared to [18F]3 but also a higher absolute tumor uptake compared to 68Ga-DOTATATE and furthermore enables an excellent tumor visualization ability combined with high image resolution but also a slowed clearance compared to 68Ga-DOTATATE. These results emphasize, emphasizing the significance importance to systematically study of the influence of molecular design and applied structure elements of peptidic radiotracers on in vivo pharmacokinetics as these may considerably influence in vivo pharmacokinetics while not affecting although not influencing other parameters such as radiochemistry, lipophilicity, serum stability or receptor binding potential.

INTRODUCTION Positron Emission Tomography (PET), an established and highly sensitive non-invasive imaging modality in clinical diagnostics, enables the visualization and quantification of complex biochemical processes. As functional imaging modality, PET allows the investigation of the in vivo distribution of radiolabeled biomolecules which ideally accumulate specifically in target tissues. Within the group of radiolabeled substances, peptides have gained high interest as radiotracers in nuclear medicine as they enable the specific visualization of target tissues such as tumors of various origins with high tumor-tobackground-ratios.1-4 For the specific PET imaging of neuroendocrine tumors (NETs) often overexpressing somatostatin receptors (SSTRs), derivatives of the endogenous peptide somatostatin have a high clinical relevance for PET imaging. For this purpose, mostly such as Tyr3-octreotate (TATE) and octreotide analogs,5, 6 have proven to be of high clinical relevance for PET imaging when radiolabeled with positron emitters such as gallium-68 (68Ga) or fluorine-18 (18F).7, 8 have proven to be highly potent PET imaging agents. Although mainly 68Ga-labeled octreotide and octreotate derivatives such as 68Ga-DOTATOC, 68 Ga-DOTANOC and 68Ga-DOTATATE (DOTA = 1,4,7,10-tetra-azacyclododecane1,4,7,10-tetraacetic acid; TOC = Tyr3-octreotide; NOC = 1Nal3-octreotide; TATE = Tyr3octreotate) are applied in clinical NET imaging with PET, also 18F-labeled analogs of these peptides are of interest. This is attributable due to the longer half-life of 18F (t1/2 = 109.7 min) compared to 68Ga (t1/2 = 68 min) and the lower mean positron energy of the emitted β+particles of 18F (249.3 keV) compared to 68Ga (836.0 keV). This lower positron energy , resultsing in better-resolved PET images in case of theusing 18F-labeled substances which is of special interest for the detection of small tumor lesions.9 However, only few 18F-radiolabeled peptides have been described for a routine clinical application due to the often cumbersome, time-consuming and inefficient multi-step 2


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radionuclide introduction procedures associated with 18F-peptide-labeling especially when compared to 68Ga.10, 11 Therefore, new 18F-labeling methods for this substance class have been developed over the last decade and are mainly based on silicon-,12, 13 boron-14, 15 and aluminium-18F-chemistry.16-18 These new 18F-labeling strategies enable a much more efficient peptide-18F-labeling radiochemistry with short reaction times, only one or very few synthesis steps and simple product workup procedures and therefore make 18F-labeled peptides easily accessible for an in vivo application. The Silicon-Fluoride-Acceptor (SiFA) 18F-labeling strategy e.g. has attracted high attention due to the strong nature of the Si-18F bond and more important its fast and efficient 18Fradiolabeling as well as the simple product workup, enabling a kit-like synthesis of 18Flabeled peptides.19-25 Its characteristics such as the simple one-step 19F-18F-isotopic exchange reaction under very mild labeling conditions (5 min at ambient temperature) producing no side-products, the small precursor amounts (nmol range) required as well as high achievable radiochemical yields and specific activities of the obtained radiolabeled compounds illustrate the advantages of this 18F-labeling chemistry. However, a structural prerequisite for a high hydrolytic stability of the 18F-Si bond are the aromatic and two tert-butyl groups on the silicon atom (Figure 1A), considerably increasing the overall lipophilicity of SiFA-modified substances. This results – if not outbalanced – in unfavorable in vivo pharmacokinetics. This as it was e.g. shown for an initially developed SiFA-TATE derivative (Figure 1C)26 and other accordingly designed 18F-labeled peptides.27 However, the high lipophilicity of the respectively derivatized radiopeptide can be compensated by the introduction of hydrophilic auxiliaries (Figure 1D and 1E) such as carbohydrates, polyethylene glycols (PEGs) or a permanently positively charged SiFA-synthon (SiFAlin, Figure 1B), resulting in improved in vivo pharmacokinetics of the so-modified radiopeptides.26, 28 By this, iIt could be shown that using the 18F-SiFA-peptide-labeling approach, a high hydrophilicity was the key parameter for achieving high tumor uptakes and favorable in vivo pharmacokinetics with a predominant renal clearance.28 In the present study, we intended to systematically evaluate the influence of the molecular design and used molecular scaffolds of SiFAlin-TATE derivatives on SSTR binding as well as on the in vivo pharmacokinetics of the resulting 18F-labeled peptides. By introducing varying numbers of negatively charged aspartic acids into the peptide sequence and replacing the so-far used non-physiologic carbohydrate moiety, In the present study, we intended meant to determine if the favorable pharmacokinetic properties of SiFAlin-TATE derivatives can be further improved by introducing a higher number of charged hydrophilic aspartic acid auxiliaries into the peptide sequence and if it is possible to replace the previously applied glucose derivative building block (Figure 1E) by physiological aspartic acids without changing the favorable in vivo pharmacokinetics of this SSTR-specific radiotracer. Therefore, new SiFAlin-TATE derivatives (Figure 1F), exhibiting the outlined molecular design, were synthesized, radiolabeled with 18F and evaluated in vitro as to their serum stability, lipophilicity and somatostatin receptor (SSTR)-specific binding potential. Furthermore, also the in vivo pharmacokinetic properties of the most promising newly developed radiotracer and thus its applicability in the PET imaging of SSTR-overexpressing tumors were assessed and 3


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compared to the so far most promising SiFAlin-TATE derivative SiFAlin-Asn(AcNH-β-Glc)Asp2-PEG1-TATE 3 (Figure 1E)28 as well as to the current clinical gold standard 68GaDOTATATE.

Fig. 1:

Structures of the Silicon-Fluoride-Acceptor (SiFA)-synthon (A), the permanently positively charged SiFAlin-synthon (B) as well as the SiFA- and SiFAlin-modified somatostatin analogs 1 − 7 (C – F).

4


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RESULTS AND DISCUSSION We intended in the present study to determine if the favorable pharmacokinetic properties of SiFAlin-TATE derivatives for the visualization of SSTR-positive tumors get influenced or can be further improved compared to previously developed analogs (such as SiFAlinAsn(AcNH-β-Glc)-Asp2-PEG1-TATE 3 (Figure 1E))28 by changing the underlying molecular design. In particular, it should be investigated if the favorable in vivo pharmacokinetics of this SiFAlin-TATE analog can be further improved by exchanging the non-physiologic carbohydrate building block by physiologic amino acids or by further increasing the hydrophilicity of the system. Radiolabeled peptides exhibiting an overall positive charge under physiological conditions can exhibit increased kidney uptakes,29-32 most probably caused by an interaction with the negatively charged extracellular domain megalin/cubilin system. In contrast, it could be shown for a bombesin analog exhibiting an overall negative charge that a radiopeptide can profit from the introduction of negatively charged amino acids in terms of in vivo pharmacokinetics compared to its neutral counterpart.32 Thus, we decided to introduce varying numbers of negatively charged and thus hydrophilicity-enhancing aspartic acids into the SiFAlin-TATE peptide sequence and also to replace the so-far used non-physiologic carbohydrate moiety. By this approach, we intended to systematically evaluate the influence of the molecular design and used molecular scaffolds of SiFAlin-TATE derivatives on SSTR binding as well as on the in vivo pharmacokinetics of the resulting 18Flabeled peptides. With these investigations, that particular 18F-labeled TATE analog should be determined which exhibits optimized properties such as high hydrophilicity, few nonphysiologic structure elements, a strong SSTR binding and most favorable in vivo pharmacokinetics for a following clinical application. introducing a higher number of charged hydrophilic aspartic acid auxiliaries and replacing the so far applied glucose derivative building block (Figure 1E) by physiological aspartic acids. Thus, the first step on this way was the synthesis of the new SiFAlin-based TATE derivatives . Synthesis of the New SiFAlin-Based TATE Derivatives 4 – 7 The preparation of the new TATE derivatives started with the synthesis of Tyr3-octreotate on solid support by standard Fmoc-based solid phase peptide synthesis (SPPS) protocols33 and successive conjugation of the Fmoc-protected amino acids applying standard coupling conditions. Further on, the peptide chain was elongated by reacting with Fmoc-NH-PEG1COOH, Fmoc-Asp(OtBu)-OH (1, 2, 3 or 4 units) and bisBoc-aminooxyacetic acid (bisBocAoa-OH) under the same reaction conditions. The intermediate products, Aoa-(Asp)n-PEG1TATE (where n = 1 – 4) 8 – 11 (Scheme 1), were isolated after cleavage from the resin and simultaneous deprotection, semipreparative HPLC purification and lyophilization in adequate overall isolated yields of 13 – 22%. For the introduction of the SiFAlin moiety, each peptide was reacted via the chemoselective oxime formation with a 5-fold excess of N-(4-(di-tert-butylfluorosilyl)benzyl)-N,N-dimethyl4-oxobutan-1-aminium bromide (SiFAlin-A 12, Scheme 1) in aqueous solution at ambient temperature for 15 minutes. The SiFAlin-modified products, SiFAlin-(Asp)n-PEG1-TATE (where n = 1 – 4) 4 – 7 (Scheme 1), were obtained in good isolated yields of 46 – 57% after 5



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final semipreparative HPLC purification and lyophilization. SiFAlin-Asn(AcNH-β-Glc)-Asp2PEG1-TATE 3 (Figure 1E) which used as a reference substance in the following in vitro and in vivo evaluations was synthesized as described before.28 Scheme 1:

H2N

HO O HN

Schematic depiction of the synthesis route of the SiFAlin-(Asp)n-PEG1-TATE derivatives 4 – 7a O NH

O

N H

HN O

S S

HN NH

HO

O

N N H O H

O

conjugation of 1) Fmoc-NH-PEG1-COOH 2) n x Fmoc-Asp(OtBu)-OH (n = 1 - 4) 3) bisBoc-Aoa-OH

O O H N

Tyr3-octreotate

Aspn

PEG1

A)

Fmoc

O

NBoc2

O

B)

OH

TFA/TIS/H2O

Wang-(Fmoc-Tyr3-octreotate)-OH synthesized by Fmoc-SPPS Tyr3-octreotate

Aspn

PEG1

O

NH2

O 8 - 11 Br N O

C)

Si H

H2N

HO O HN

O NH

O

HO N H

HN O

S S

HN NH

O

F

12

N N H O H

O

Br

HO

N

n

O

O

H N

O O

O

O

H N

N H HO

N

Si

F

O O

n=1-4

OH 4-7

a

Reagents and Conditions: A) 4 eq. amino acid, 3.9 eq. HBTU (N,N,N′,N′-tetramethyl-O(1H-benzotriazol-1-yl)uronium hexafluoro-phosphate), 4 eq. DIPEA (N,N-diisopropylethylamine), DMF, RT, 30 min; B) TFA (trifluoroacetic acid) / TIS (triisopropylsilane) / H2O 95%/2.5%/2.5%, RT, 1 h, yields: 13 – 22% over all synthesis steps of including the SPPS of TATE A) and B); C) 5 eq. SiFAlin-A, phosphate buffer (0.5 M, pH 4.0)/MeCN, RT, 15 min, yields: 46 – 57%.

18

F-Radiolabeling of the New SiFAlin-TATE Derivatives 4 – 7 The SiFAlin-TATE modified peptides 4 – 7 were in the following successfully radiolabeled with [18F]fluoride by applying a simple and efficient one-step 19F-by-18F-isotopic exchange reaction which takes place within 5 minutes reaction time at ambient temperature and gives no radioactive side products. The dried [18F]fluoride which was applied in the radiolabeling reactions was obtained by the “Munich [18F]fluoride drying method” using a strong anion-exchange (SAX, SepPak Accell 6


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Plus QMA carbonate light) cartridge,34 yielding a stock solution of dried [18F]F-/[K+⊂2.2.2]OH- complex in 500 µL MeCN. As the SiFAlin-synthon is susceptible to strongly basic conditions, the base in the [18F]fluoride stock solution had to be partly neutralized by the addition of oxalic acid before incubating with the SiFAlin-based labeling precursors.35 The amount of oxalic acid which is required to enable a high 18F-incorporation has to be determined experimentally for each substance and was found to be ideal for the labeling of 4 – 7 when reaching a ratio of potassium hydroxide to oxalic acid of 4:1. After partial neutralization of the [18F]fluoride solution, 25 nmol of the respective precursor peptide were added and after 5 minutes reaction time, the mixtures were analyzed by analytical radio-HPLC, showing 18F-incorporation rates of 90% to 97%. Due to these highly efficient labeling reactions giving no side products and as the precursor molecules and 18Flabeled products are chemically identical due to the isotopic exchange reaction, only unreacted [18F]fluoride has to be removed after labeling. This is accomplished by applying a simple C18 cartridge-based purification step. After cartridge purification, the 18F-labeled products [18F]4 – [18F]7 could be obtained in high radiochemical yields (RCYs) and purities (RCPs) of 70 – 80% and ≥97%, respectively. The non-optimized specific activities (SAs) ranged from 20.1 to 45.2 GBq/µmol, starting from 0.7 – 1.5 GBq of the dried [18F]F/[K+⊂2.2.2]OH- complex, and were thus sufficiently high for in vivo PET/CT tumor imaging. This unpretentious experimental setup requiring no sophisticated manipulation or purification steps further enables a kit-like formulation of the respective 18F-labeled peptides which is highly advantageous for a transfer of this radiotracer to a clinical application. Figure 2 schematically depicts the steps of the 18F-labeling reaction of [18F]4 – [18F]7. [18F]3 was radiolabeled as described before28 and used as a reference substance in the following in vitro and in vivo evaluations.

7



step 1 drying of [18F]F(Munich method)

QMA Carbonate cartridge-based drying [18F]F-/[K+⊂2.2.2]OH- complex

in ~ 500 µL MeCN analytical radio-HPLC chromatograms of crude products 1000

18

[ F]4 18 [ F]5 18 [ F]6 18 [ F]7

20

[18F]F800

step 2

10

19F-18F-isotopic cps

600

exchange reaction

0 1

2

400

200

18F-incorporation:

90 – 97% 0 0

1

2

3

4

5

time [min]

analytical radio-HPLC chromatograms of purified products

1000

18

[ F]4 18 [ F]5 18 [ F]6 18 [ F]7

C18 cartridge purification,

step 3 purification of labeled peptides

product obtained in 90% ethanol/10%H2O

800

600

RCPs: ≥97% cps

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RCYs: 70 – 80% SAs: 20.1 – 45.2 GBq/µmol (start: 0.7 – 1.5 GBq)

400

200

0 0

1

2

3

4

5

time [min]

Fig. 2:

Schematic depiction of the radiosynthesis steps of the TATE derivatives [18F]4 – [18F]7.

18

F-SiFAlin-(Asp)n-PEG1-

In Vitro Evaluations of the Newly Developed Substances [18F]4 – [18F]7 in Comparison to Reference [18F]3. Determination of the Stability of [18F]4 – [18F]7 in Human Serum. In order to ensure a sufficient stability of the 18F-labeled peptides for to justify an in vivo application as it was shown before for [18F]3,28 their stability was determined in human serum. For this purpose, an aliquot of the respective radiotracer solution containing 71 – 133 MBq of the cartridge-purified product [18F]4 – [18F]7 was added to 500 µL of human serum and incubated at 37°C for 90 minutes. At certain time points of 0, 5, 10, 20, 30, 40, 60 and 90 minutes, aliquots of 75 µL were taken, serum proteins were precipitated with MeCN, precipitate and supernatant were measured for radioactivity and the supernatant was analyzed by analytical radio-HPLC. These analyses showed no degradation of the radiolabeled peptides over time (Figure S1). Thus, these results indicate a suitable stability of the developed SiFAlin-based TATE derivatives [18F]4 – [18F]7 exhibit a suitable stability for in vivo imaging studies. These found high serum stabilities are se results are in accordance with results of previous studies also showing a high serum stability of SiFA- and SiFAlin-based peptidic 8


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radiotracersdata published for in vitro stability of several SiFA-derivatized peptides findings. 26, 28

Determination of the Lipophilicity of [18F]4 – [18F]7 and in comparison to [18F]3. We and others showed before that a low lipophilicity of peptidic radiotracers is a mandatory prerequisite Since for a successful in vivo tumor imaging with PET as otherwise, a high liver accumulation and low tumor uptakes can be observed.27, 28 However, we were also able to demonstrate that by increasing the hydrophilicity of SiFA-based TATE derivatives, high tumor accumulations and low background activity levels can be achieved. Here we intended to evaluate that of the newly developed radiopeptides [18F]4 – [18F]7 in vivo which showed a comparable hydrophilicity than reference [18F]3 and comprised the lowest number of aspartic acids. For this purpose, the lipophilicity of [18F]4 – [18F]7 and [18F]3 was comparatively evaluated by a partition experiment, incubating the radiolabeled peptides (5.2 – 7.7 MBq) with a mixture of aqueous phosphate buffer (pH 7.4) and 1-octanol for 5 minutes. After separation of aqueous and organic phase, the activity accumulated in both phases was measured and the lipophilicity of the radiolabeled peptides calculated. Table 1 shows the results of these experiments. Table 1: Lipophilicities (LogD) of the 18F-SiFA- and 18F-SiFAlin-modified TATE derivatives as determined by 1-octanol/water partition experiments (n = 6).

18

F-SiFA- or 18F-SiFAlin-TATE derivative

LogD

SiFA-TATE, 1

1.59 ± 0.0126

SiFA-Asn(AcNH-β-Glc)-PEG1-TATE, 2

0.96 ± 0.0726

18

F-SiFAlin-Asn(AcNH-β-Glc)-Asp2-PEG1-TATE, [18F]3

-1.21 ± 0.0728 -1.29 ± 0.03

18

F-SiFAlin-Asp1-PEG1-TATE, [18F]4

-0.04 ± 0.03

18


-1.01 ± 0.02

18

F-SiFAlin-Asp3-PEG1-TATE, [ F]6

-1.23 ± 0.03

18


-1.50 ± 0.04

18

Field Code Changed Field Code Changed

As expected, it was found that the lipophilicity of the new radiopeptides decreased with increasing number of aspartic acids introduced into the peptide sequence of from [18F]4 – [18F]7. As can be further inferred from these data, a significantly reduced lipophilicity was found for the newly developed SiFAlin-(Asp)n-PEG1-TATE conjugates 4 – 7 compared to the former SiFA-based TATE derivatives SiFA-TATE 1 and SiFA-Asn(AcNH-β-Glc)-PEG1TATE 2. Furthermore, the introduction of aspartic acids could outbalance the high lipophilicity of the SiFAlin-synthon just as good well as the glucose-based auxiliary which was used before in SiFAlin-Asn(AcNH-β-Glc)-Asp2-PEG1-TATE [18F]3,. rendering 9



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especially 6 and 7 well-suited for in vivo imaging applications due to their expected renal clearance. As [18F]6 and [18F]7 exhibit a comparable or even higher hydrophilicity than [18F]3, these two radiopeptides should be suitable for an in vivo imaging application due toregarding their expected predominant renal clearance.

Determination of the SSTR-Binding Potential of 4 – 7 and 3 on AR42J Tumor Cells. In the following, the SSTR-specific binding affinities of 4 – 7 and reference 3 were evaluated by a competitive displacement assay on viable, SSTR-expressing36 rat pancreas carcinoma AR42J tumor cells with 177Lu-DOTATATE as the competing radioligand.28 In Figure 3, the results of this assay are summarized in form of the obtained binding curves and calculated IC50 values. The results show a high strong and specific binding of all tested compounds to the SSTR-positive cells. As expected from earlier studies, showing only a minor influence of various N-terminal modifications on the resulting peptide-SSTR binding affinities (regardless of size and complexity of the chosen modification),26, 30, 37 the number of introduced aspartic acids had no observable influence on the found binding affinities to AR42J tumor cells.

3 (IC50: 14.4 ± 1.2 nM)

100

4 (IC50: 17.9 ± 1.1 nM) 5 (IC50: 18.0 ± 0.8 nM) 6 (IC50: 20.7 ± 2.5 nM) 7 (IC50: 19.7 ± 0.5 nM)

80 60 40 20 0 -10

-9

-8

-7

-6

log c

Fig. 3:

Results of the competitive displacement assay for 4 − 7 and 3 as obtained by three independent experiments each performed in triplicate.

Taking these in vitro evaluation data into account, it was anticipated that [18F]6 should be the most potent of the newly developed substances for the following in vivo evaluation as: i) the newly developed substances 4 − 7 all showed a comparable SSTR binding potential, not singling one substance out as particularly potent, ii) [18F]6 showed a comparable hydrophilicity to the reference substance [18F]3 which exhibited a favorable pronounced renal clearance in vivo28 and as iii) we anticipated that further increasing the number of aspartic acids from three ([18F]6) to four ([18F]7) a further increased number of negative charges as in case of [18F]7 might result 10


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in a higher kidney accumulation and thus lower tumor-to-organ ratios.30, 38 Thus, we intended to start the in vivo evaluations of the newly developed tracers with radiopeptide [18F]6. In case of superior pharmacokinetics of [18F]6 compared to [18F]3, the subsequent evaluation of the in vivo pharmacokinetic profile of [18F]7 would have been performed as well. Due to these considerations, we first evaluated in a decelerated clearance and higher unspecific and thus a lower tumor accumulation of the radiotracer. Thus, [18F]6 was further evaluated in vivo in AR42J tumor-bearing mice.

Evaluation of the In Vivo Pharmacokinetics of [18F]6 in AR42J Tumor-Bearing Mice in Comparison to [18F]3 and 68Ga-DOTATATE as Reference Substances. To investigate the in vivo pharmacokinetic properties of [18F]6, small-animal PET/CT imaging studies were performed in tumor-bearing nude mice exhibiting a subcutaneous SSTR-expressing AR42J tumor. For the imaging study, 5.2 ± 1.1 MBq of [18F]6 were administered via the lateral tail vein under isoflurane anesthesia. As reference substances, [18F]3 and the clinical gold standard 68Ga-DOTATATE were evaluated under the same conditions. The results of these in vivo PET/CT imaging studies are shown in Figure 4 as well as in Table 2, Figures S2 and S3.

Fig. 4:

Small animal PET/CT imaging results for [18F]6, [18F]3 and 68Ga-DOTATATE in AR42J tumor-bearing mice. The images show maximum intensity projections for all tracers over 90 minutes post injection and give the found mean SUVs in the tumor tissues. (A) Shows the pharmacokinetics of [18F]6 (n = 6) and the corresponding blocking experiment (n = 5), (B) and (C) show the pharmacokinetics of the reference substances [18F]3 (n = 5) and 68Ga-DOTATATE (n = 4), respectively.

11



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Table 2: Tumor-to-organ-ratios for [18F]6, [18F]3 and points as obtained from in vivo PET data. [18F]6

68

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Ga-DOTATATE at different time-

68

[18F]3

Ga-DOTATATE

time [min]

T/M

T/B

T/L

T/K

T/M

T/B

T/L

T/K

T/M

T/B

T/L

T/K

32.5

17.31 ± 4.52

4.54 ± 1.23

3.42 ± 0.80

0.71 ± 0.18

22.98 ± 8.79

6.64 ± 2.28

6.00 ± 1.87

0.95 ± 0.29

25.92 ± 7.44

5.40 ± 2.34

2.79 ± 1.24

1.58 ± 0.93

65

25.50 ± 9.13

6.87 ± 2.17

4.85 ± 1.37

0.92 ± 0.29

39.35 ± 14.57

11.74 ± 4.66

8.97 ± 2.88

1.21 ± 0.37

61.57 ± 21.28

10.23 ± 4.72

3.70 ± 1.73

2.29 ± 1.41

85

29.56 ± 11.33

8.06 ± 2.72

5.42 ± 1.61

0.99 ± 0.29

49.27 ± 20.50

14.96 ± 5.99

10.35 ± 3.28

1.26 ± 0.39

91.97 ± 45.13

13.41 ± 6.59

4.03 ± 1.91

2.41 ± 1.65

T/M = tumor-to-muscle-ratio; T/B = tumor-to-blood-ratio; T/L = tumor-to-liver-ratio; T/K = tumor-to-kidney-ratio

Compared to the clinically established gold standard 68Ga-DOTATATE for PET imaging of SSTR-positive tumors, both 18F-SiFAlin-TATE derivatives [18F]6 and [18F]3 showed a significantlyn absolute higher absolute tumor uptake than 68Ga-DOTATATE (Figure 4 and S3)maximum SUVs of 6.94 ± 1.61 in case of [18F]6 (Figure 4A), 7.70 ± 2.17 in case of [18F]3 (Figure 4B) and 4.34 ± 1.50 in case of 68Ga-DOTATATE (Figure 4C)) over 90 minutes. The SSTR-specificity of the tumor uptake of [18F]6 was confirmed by a blocking experiment (Figure 4A). [18F]6 furthermore showed, an excellent stability over the course of imaging and as expected from the lipophilicity data, the a retained favored renal excretion could be retained by the chosen molecular design. For both SiFAlin-TATE derivatives [18F]6 and [18F]3, a slightly higher kidney accumulation and decelerated renal excretion could be observed compared to 68Ga-DOTATATE (Figure S2), being most probably a result of the higher negative net charge of the former (net charges for 68Ga-DOTATATE: ±0, [18F]3: -1 and [18F]6: -2). This effect of charge-induced retention is also reflected supported byin the more pronounced kidney uptake of [18F]6 compared to [18F]3 which should be attributable to the additional negative charge of the introduced aspartic acid and isto some extent lower tumor-to-organ-ratios which were observable for [18F]6 and [18F]3 compared to 68Ga-DOTATATE (Table 2) in accordance with results of previous studies which also found higher kidney uptakes and retention of negatively charged radiopeptides compared to non-charged ones.30, 38, 39 This is also reflected in the to some extent lower tumor-to-organ-ratios which were observable for [18F]6 and [18F]3 compared to 68 Ga-DOTATATE (Table 2). Nevertheless, [18F]6 and [18F]3 both enabled an excellent tumor visualization (attributable due to the very high tumor accumulation of the radioligands (Figure S2). Furthermore, the 18F-labeled TATE analogs enabled a significantly improved ) and image resolution attributable to the lower β+ energy in case of 18F compared to 68Ga-DOTATATE (Figure 4) being favorable for the detection of even small tumors and metastases. When directly cComparing the in vivo pharmacokinetics and especially the tumor-to-organratios of [18F]6 and [18F]3, it becomes clear that [18F]6 is stronger retained taken up to a somewhat higher extent in non-target tissues and is longer retained in the kidneys (Figure S2), thus resulting in better imaging results for [18F]3. This indicates that the introduction of hydrophilic, but uncharged carbohydrate auxiliaries seems to be preferable over charged 12


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aspartic acids when intending to reduce the overall lipophilicity of a radiopeptide as the introduction of charges does not necessarily influence radiochemistry, serum stability or receptor binding potential but can impair in vivo pharmacokinetics of a respectively modified radiopeptide. Thus, [18F]3 shows the highest potential for the imaging of SSTR-positive neuroendocrine tumors and a further clinical evaluation.

CONCLUSION We could show here that by the introduction of hydrophilic aspartic acid moieties, the lipophilicity hydrophilicity of SiFAlin-modified peptidic radioligands can efficiently be reduced increased without negatively affecting radiolabeling, serum stability or the high and specific in vitro receptor binding affinity. In contrast, a considerable influence of the introduced negative charges on the in vivo radiotracer pharmacokinetics was observed and resulted in higher unspecific organ uptakes slightly lower tumor-to-background ratios of the SiFAlin-TATE derivative comprising a higher number of aspartic acids compared to the carbohydrate-modified one. This effect can be attributed to a slightly decelerated blood clearance and a higher kidney uptake of the highly-charged radioligand. Nevertheless, the developed somatostatin analog 18F-SiFAlin-(Asp)3-PEG1-TATE exhibited high absolute tumor accumulations and furthermore enabled an excellent tumor visualization ability and image quality in PET/CT imaging. These results indicate that the introduction of hydrophilic, but uncharged carbohydrate auxiliaries seems to be preferable over charged aspartic acids in terms of in vivo pharmacokinetics although not influencing other parameters such as radiochemistry, serum stability or receptor binding potential of the peptidic radiotracers.

EXPERIMENTAL SECTION General. All commercially available chemicals were of analytical grade and used without further purification. The resin (Fmoc-Thr(tBu)-Wang resin), Fmoc-protected amino acids, Fmoc-NH-PEG1-COOH and bisBoc-Aoa-OH for Fmoc solid-phase peptide synthesis were obtained from Merck Millipore. Tris-tBu-DOTA and HEPES (4-(2-hydroxyethyl)piperazine1-ethanesulfonic acid) were purchased from CheMatech and GERBU, respectively. Water for 18 F-labeling (Tracepur quality) and Kryptofix 2.2.2 were obtained from Merck. [18F]fluoride was obtained from the Zyklotron AG Karlsruhe, Germany and 177Lu-DOTATATE used for competitive displacement experiments was obtained from the University Hospital Mainz, Department of Nuclear Medicine, Germany. SepPak Accell Plus QMA Carbonate light and C18 light cartridges were obtained from Waters. 3 and [18F]3 were synthesized as described before.28 General information about used materials and instruments, details concerning peptide syntheses, PET data analyses, the determination of serum stabilities and lipophilicities of [18F]4 – [18F]7 and cell culture can be found in the supplemental information. Also, serum 13



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stability data of [18F]4 − [18F]7 in human serum and in vivo pharmacokinetic data of [18F]6, [18F]3 and 68Ga-DOTATE can be found in the supplemental information. AR42J rat pancreatic tumor cells were obtained from the European Collection of Cell Cultures (ECACC). RPMI 1640 medium, 200 mM L-glutamine and 0.05% trypsinethylenediaminetetraacetic acid (trypsin-EDTA (phenol red)) were purchased from Gibco (Life technologies). Fetal calf serum (FCS), Dulbecco’s phosphate buffered saline (PBS) and protease inhibitor cocktail were obtained from GE Healthcare (PAA) and Sigma Aldrich, respectively. Bovine serum albumin fraction V (BSA) was purchased from Carl Roth. Magnesium chloride hexahydrate (MgCl2 ⨉ 6H2O) and calcium chloride dihydrate (CaCl2 ⨉ 2H2O) were supplied by VWR and Merck. For analytical, semipreparative and radio-HPLC chromatography, a Dionex UltiMate 3000 system (Thermo Fisher Scientific) was used, equipped with a Chromolith Performance column (RP-18e, 100 – 4.6 mm, Merck, Germany), a semipreparative Chromolith column (RP-18e, 100 – 10 mm, Merck, Germany) as well as a Raytest Gabi Star radioactivity detector and operated with a flow of 4 mL/min. MALDI (Matrix-Assisted Laser Desorption/Ionization) spectra were obtained using a Bruker Daltonics Microflex spectrometer. The gamma-counter used was a 2480 WIZARD2 (PerkinElmer). The smallanimal PET/CT imaging studies were performed on a Bruker Albira PET/SPECT/CT system. Synthesis of the Aoa-(Asp)n-PEG1-TATE Derivatives 8 – 11. Tyr3-octreotate (0.05 or 0.1 mmol) was synthesized using standard reactions conditions (see supplemental information for details). according to a published standard Fmoc-based solid phase peptide synthesis (SPPS) approach29 on a Fmoc-Thr(tBu)-Wang resin (coupling conditions: 4 eq. amino acid, 3.9 eq. HBTU (N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluoro-phosphate), 4 eq. DIPEA (N,N-diisopropylethylamine) in DMF for 30 minutes; Fmoc cleavage: 50% piperidine in DMF). After cyclization of the Tyr3-octreotate peptide sequence by incubating the resin with Tl(III)-trifluoroacetate (4 eq.) in DMF for 45 minutes, the terminal Fmoc protecting group was removed and the respective hydrophilic auxiliaries Fmoc-NH-PEG1-COOH, FmocAsp(OtBu)-OH and bisBoc-Aoa-OH were successively conjugated using the same coupling conditions. Subsequently, the modified TATE derivatives were cleaved from the resin using a mixture of TFA:TIS:H2O (95:2.5:2.5) for 60 minutes, precipitated in diethyl ether, dried and purified by semipreparative HPLC. The products were isolated as white solids after lyophilization. Gradients used for HPLC purification, synthesis yields and analytic data for each compound are given below. 8: gradient: 0–40% MeCN (acetonitrile) + 0.1% TFA in 8 min, yield: 22.3% (15.4 mg, 11.1 µmol), MALDI-MS (m/z) for [M+H]+ (calculated): 1382.66 (1382.52), (m/z) for [M+Na]+ (calculated): 1404.75 (1404.42), (m/z) for [M+K]+ (calculated): 1420.72 (1420.62). 9: gradient: 0–40% MeCN + 0.1% TFA in 8 min, yield: 19.0% (28.5 mg, 19.0 µmol), MALDI-MS (m/z) for [M+H]+ (calculated): 1497.35 (1497.56), (m/z) for [M+Na]+ (calculated): 1519.36 (1519.55), (m/z) for [M+K]+ (calculated): 1535.36 (1535.66). 10: gradient: 0–40% MeCN + 0.1% TFA in 8 min, yield: 17.5% (28.2 mg, 17.5 µmol), MALDIMS (m/z) for [M+H]+ (calculated): 1612.92 (1612.59), (m/z) for [M+Na]+ (calculated): 1634.99 (1634.58), (m/z) for [M+K]+ (calculated): 1651.01 (1650.69). 11: gradient: 0–40% MeCN + 0.1% TFA in 8 min, yield: 12.8% (11.1 mg, 6.42 µmol), MALDI-MS (m/z) for 14


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[M+H]+ (calculated): 1727.62 (1727.61), (m/z) for [M+Na]+ (calculated): 1749.66 (1749.51), (m/z) for [M+K]+ (calculated): 1765.55 (1765.71). Synthesis of N-(4-(di-tert-butylfluorosilyl)benzyl)-N,N-dimethyl-4-oxobutan-1ammonium Bromide (SiFAlin-A, 12). To N-(4-(di-tert-butylfluorosilyl)benzyl)-4,4diethoxy-N,N-dimethylbutan-1-ammonium bromide (30.0 mg, 57.6 µmol), which was synthesized as described before28, was added an aqueous TFA solution (95% (v/v) TFA, 100 µL) and the reaction was stirred at ambient temperature for 30 minutes. The solution was concentrated in vacuo and the product was precipitated by addition of 1.5 mL diethyl ether at -28°C overnight. The precipitate was isolated by centrifugation and decantation, yielding 12 as white solid which was directly used for the subsequent conjugation to the aminooxymodified TATE derivatives 8 – 11 without further purification. Synthesis of the SiFAlin-(Asp)n-PEG1-TATE Derivatives 4 – 7. To a solution of the Aoa(Asp)n-PEG1-TATE derivatives 8 – 11 (11.1 – 16.8 mg, 6.42 – 11.2 µmol) in phosphate buffer (0.5 M, pH 4.4, 200 µL) was added a solution of 8 (5 eq., 14.3 – 25.1 mg, 32.1 – 56.1 µmol) in acetonitrile (200 µL). The mixture was allowed to react for 15 minutes before the products were purified by semipreparative HPLC and isolated as white solids after lyophilization. Gradients used for HPLC purification, synthesis yields and analytic data for each compound are given below. 4: gradient: 10–20% MeCN + 0.1% TFA in 1 min, followed by 20–70% MeCN + 0.1% TFA in 7 min, yield: 50.8% (9.8 mg, 5.66 µmol), MALDI-MS (m/z) for [M+H]+ (calculated): 1730.45 (1730.78), (m/z) for [M+Na]+ (calculated): 1752.42 (1752.77), (m/z) for [M+K]+ (calculated): 1768.48 (1768.88). 5: gradient: 10–20% MeCN + 0.1% TFA in 1 min, followed by 20–70% MeCN + 0.1% TFA in 7 min, yield: 46.4% (9.6 mg, 5.20 µmol), MALDI-MS (m/z) for [M+H]+ (calculated): 1845.23 (1845.81), (m/z) for [M+Na]+ (calculated): 1866.88 (1867.80), (m/z) for [M+K]+ (calculated): 1883.21 (1883.91). 6: gradient: 10–20% MeCN + 0.1% TFA in 1 min, followed by 20–70% MeCN + 0.1% TFA in 7 min, yield: 46.3% (8.5 mg, 4.33 µmol), MALDI-MS (m/z) for [M+H]+ (calculated): 1960.85 (1960.84), (m/z) for [M+Na]+ (calculated): 1982.57 (1982.83), (m/z) for [M+K]+ (calculated): 1998.21 (1998.94). 7: gradient: 10–20% MeCN + 0.1% TFA in 1 min, followed by 20–70% MeCN + 0.1% TFA in 7 min, yield: 57.0% (7.6 mg, 3.66 µmol), MALDI-MS (m/z) for [M+H]+ (calculated): 2074.66 (2075.86), (m/z) for [M+Na]+ (calculated): 2096.51 (2097.85), (m/z) for [M+K]+ (calculated): 2112.84 (2113.96). Synthesis of DOTATATE. Tyr3-octreotate (0.1 mmol) was obtained by standard Fmoc SPPS as described previously and further reacted with tris-tBu-DOTA (coupling conditions: 2.5 eq. chelator, 2.45 eq. HBTU and 5 eq. DIPEA in DMF) for 1.5 hours. The product was cleaved from the resin using a mixture of TFA:TIS:H2O (95:2.5:2.5) for 5 hours, precipitated in diethyl ether, dried and purified by semipreparative HPLC. The product was isolated as white solid after lyophilization. The gradient used for HPLC purification, synthesis yields and analytic data are given below. DOTA-TATE: gradient: 0–40% MeCN + 0.1% TFA in 8 min, yield: 21.2% (30.5 mg, 21.2 µmol), MALDI-MS (m/z) for [M+H]+ (calculated): 1436.32 (1435.59), (m/z) for 15



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[M+Na]+ (calculated): 1458.46 (1457.58), (m/z) for [M+K]+ (calculated): 1474.39 (1473.69), (m/z) for [M+Na+K]+ (calculated): 1496.37 (1495.68). Radiosynthesis of [18F]4 – [18F]7. Preparation of [18F]fluoride by the Munich method.34 The aqueous [18F]fluoride solution was loaded on a SepPak Accell Plus QMA Carbonate light cartridge (46 mg) preconditioned with 10 mL Tracepur H2O. After washing the cartridge with 5 mL anhydrous acetonitrile to remove traces of water and subsequent drying with 20 mL air, the [18F]fluoride was eluted from the cartridge with a solution of Kryptofix 2.2.2 (41 mg, 110 µmol) and potassium hydroxide (1 M, 100 µL, 100 µmol) in anhydrous acetonitrile (500 µL). The obtained dried [18F]F-/[K+⊂2.2.2]OH- complex (in 450–500 µL MeCN) was directly used for the subsequent radiolabeling of the precursor peptides 4 – 7. Radiolabeling of 4 – 7. To the dried [18F]F-/[K+⊂2.2.2]OH- complex (in 450 – 500 µL MeCN) – obtained as described before – was first added a solution of H2C2O4 (1 M, 25 µmol, 25 µL) in anhydrous acetonitrile and afterwards a solution of the respective peptidic labeling precursor 4 – 7 (25 nmol) in a mixture of anhydrous MeCN and DMSO (1:1, 50 µL). After 5 minutes at ambient temperature, the reaction mixture was analyzed by analytical radio-HPLC. The obtained product solution was quickly added to HEPES (4-(2-hydroxyethyl)piperazine-1ethanesulfonic acid) buffer (0.1 M, pH 2.0, 9 mL) and loaded on a SepPak C18 light cartridge preconditioned with 5 mL ethanol, followed by 10 mL Tracepur H2O. The cartridge was washed with phosphate buffer (0.05 M, pH 7.4, 9 mL) and Tracepur H2O (1 mL) to remove traces of unreacted [18F]fluoride and afterwards dried with 5 mL of air. The products [18F]4 – [18F]7 were eluted from the cartridge with a mixture of ethanol and Tracepur H2O (9:1, 500 µL). The obtained ethanolic solution was diluted with isotonic saline solution to give a final concentration (v/v) of ethanol of maximal 10%. The radiochemical purity of the products was determined by analytical radio-HPLC (conditions: 0–100% MeCN + 0.1% TFA in 5 min, unreacted 18F-fluoride elutes at 0.6 min (free 18F-fluoride) and 0.9 min (Kryptofix-18F-fluoride complex) under these conditions) and confirmed to be ≥97%. The overall synthesis time was 20 – 25 minutes. The radiolabeled peptides [18F]4 – [18F]7 were obtained in radiochemical yields of 70 – 80% (decay corrected to start of synthesis) and non-optimized specific activities of 20.1 – 45.2 GBq/µmol, starting from 0.7 –1.5 GBq of the [18F]F-/[K+⊂2.2.2]OH- complex. Determination of Serum Stabilities of [18F]4 – [18F]7. A solution of the respective radiolabeled peptide [18F]4 – [18F]7 (55 µL, 71 – 133 MBq) – obtained as described before – was added to 500 µL of human serum and incubated at 37°C. After 5, 10, 20, 30, 40, 60 and 90 minutes, aliquots of 75 µL were taken and added to the same volume of MeCN and the samples were stored on ice for 5 – 10 minutes to complete the precipitation of serum proteins. The precipitates were removed by centrifugation, precipitates and supernatants were measured for radioactivity and the supernatants were analyzed by analytical radio-HPLC. Determination of Lipophilicities of [18F]4 – [18F]7 and [18F]3. A solution of the respective radiolabeled peptide [18F]4 – [18F]7 or [18F]3 (2 µL, 5.2 – 7.7 MBq) obtained as described before was added to a mixture of 1-octanol (800 µL) and phosphate buffer (0.05 M, pH 7.4, 800 µL) and the resulting mixtures were vigorously vortexed for 5 minutes. After subsequent 16


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centrifugation for phase separation, aliquots of 300 µL were taken from each phase and measured for radioactivity. The LogD of the radiolabeled peptides was calculated from six independent partition experiments. Determination of SSTR Binding Affinities of 4 – 7 and 3. Cell Culture. AR42J rat pancreatic tumor cells were cultured in RPMI 1640 medium supplemented with L-glutamine (1%, 2 mM) and FCS (20%) at 37°C in a humidified incubator (5% CO2). For cell transfer and further culture, the cells were trypsinized with trypsin-EDTA (0.05%) for 3 minutes. Competitive Displacement Assay. The in vitro binding affinities of 4 – 7 and 3 were determined on viable AR42J cells via competitive displacement experiments using 177LuDOTATATE as competitor on a Millipore Multiscreen punch kit. The Millipore 96-well filter plates were incubated with 200 µL of PBS (phosphate buffered saline)/1% BSA (bovine serum albumin fraction V) solution per well for 1 hour before use. AR42J tumor cells were harvested, counted, resuspended in aqueous binding buffer (modified with 10 mM MgCl2 ⨉ 6H2O (magnesium chloride hexahydrate), 1 mM CaCl2 ⨉ 2H2O (calcium chloride dehydrate), 25 mM HEPES and 0.5% BSA, pH 7.4) together with protease inhibitor cocktail (1 : 200dilution) and seeded in 96-well filter plates at 105 cells per well. Subsequently, the cells were incubated at ambient temperature for 1 hour with 0.5 nM 177Lu-DOTATATE per well in the presence of increasing concentrations of the respective peptide 4 – 7 or 3 ranging from 0.1 to 250 nM in a total volume of 100 µL. After incubation, the reaction medium was aspirated and the cell pellets were washed three times with cold PBS (2 ⨉ 100 µL, 1 ⨉ 200µL) using the Millipore Multiscreen vacuum manifold for filtration. The radioactivity of the cells was measured in a gamma-counter. Each experiment was carried out in triplicate. The IC50 values of the peptides were calculated by non-linear regression analysis using the GraphPad Prism software (version 6). Comparative In Vivo Evaluation of [18F]6, [18F]3 and 68Ga-DOTATATE in AR42J Tumor-Bearing Mice. General. All animal experiments were performed in compliance with the German animal protection laws and protocols of the local committee. Female Crl:CD1-Foxn1nu nude mice (20 – 30 g, 12 – 13 weeks) were obtained from Charles River and subcutaneously injected with an AR42J cell suspension (5 ⨉ 106 cells per mouse, 100 µL) into the left flank under isoflurane anesthesia. The tumors were allowed to grow for 10 – 14 days and the average tumor size was 100 ± 50 mg at the time of the PET/CT imaging studies. Small-animal PET/CT imaging studies. For the imaging studies, the radiotracers [18F]6, [18F]3 and 68Ga-DOTATATE (5.2 ± 1.1 MBq) were applied via the lateral tail vein under isoflurane anesthesia. For the blocking experiments, the animals were concomitantly injected with 200 µg DOTATATE together with the radiotracer. The animals were imaged over 120 minutes (90 min for PET and 30 min for CT imaging). The PET image reconstruction was performed using the maximum likelihood expectation-maximization (MLEM) routine with options for 0.5 mm (cubic) image voxel sizes and scatter and decay correction. The acquired data sets were reconstructed using a total of 29 frames (10×60 s, 10×20 s, 6×300 s, 3×600 s). The CT images were obtained at 45 kVp, with currents of 0.4 mA (high dose, good 17



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resolution). Acquisitions of 400 projections were taken and a 250 µm isotropic voxel size image was reconstructed via filtered back projection. The images were reconstructed using the PMOD software (version 3.6). After verification of automated PET/CT coregistration, volumes of interest (VOIs) were defined by visual inspection for quantification of tracer accumulation in different tissues (tumor, heart, liver, kidneys, bladder, muscle and bone). The results for each VOI were calculated as SUVbw (g/mL) averaged for each time frame.

CORRESPONDING AUTHORS *Email: [email protected]. Phone: +49 621 383 3761. Fax: +49 621 383 1473. *Email: [email protected]. Phone: +49 621 383 5594. Fax: +49 621 383 1473. Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS The authors acknowledge financial support by the Ministry of Science, Research and the Arts of the State of Baden-Württemberg and the German Federal Ministry of Education and Research (BMBF) within the Framework “Forschungscampus: public-private partnership for Innovations” – Research Campus M²OLIE. Stephan Maus is gratefully acknowledged for providing the 177Lu-DOTATATE.

KEYWORDS 18

F, AR42J, PET imaging, SiFAlin, TATE analogs, SSTR

REFERENCES (1) (2)

Breeman, W. A. P., Kwekkeboom, D. J., de Blois, E., de Jong, M., Visser, T. J., and Krenning, E. P. (2007) Radiolabelled regulatory peptides for imaging and therapy. AntiCancer Agents Med. Chem. 7, 345-357. Wängler, C., Buchmann, I., Eisenhut, M., Haberkorn, U., and Mier, W. (2007) Radiolabeled peptides and proteins in cancer therapy. Protein Pept. Lett. 14, 273-279.

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(3) (4) (5)

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Next Generation of SiFAlin-Based TATE Derivatives for PET Imaging

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