PET and Biodistribution Experiments

The Y₁ receptor selectivity was exemplary confirmed for antagonists 5.16 and 5.17, the most potent potential PET ligands, using established flow cytometric binding assays based on fluorescence labeled pNPY and PP (Table 2).

Table 2. NPY receptor subtype selectivity of the potential PET ligands 5.16 and 5.17.

Compd. Y₁ K_i [nM]^a Y₂ K_i [nM]^b Y₄ K_i [nM]^c Y₅ K_i [nM]^b 5.16 1.3 > 2,000 > 10,000 > 5,000 5.17 7 > 2,000 > 5,000 > 5,000

aDissociation constant from radioligand competition assay with [³H]-UR-MK114 (c = 1.5 nM) on SK-N-MC neuroblastoma cells. ^bFlow cytometric binding assay on CHO-Y2

and HEC-1B-Y5 cells using Dy-635-pNPY as labeled ligand (10 nM). ^cFlow cytometric binding assay on CHO-Y4 cells with Cy5-[K⁴]-hPP (5 nM) as fluorescent ligand.

5.2.3 PET and Biodistribution Experiments

Fluorine-18 labeling of precursors 2.7 and 4.21 to obtain the Y₁R antagonistic PET ligands 5.8-F18 and 5.9-5.8-F18 (Scheme 5) was performed in the laboratory of Prof. Dr. H.J. Wester at the Nuclear Medicine Department of the Klinikum rechts der Isar, TU München. Both PET ligands were administered intravenously to male NMRI (nu/nu) mice with SK-N-MC xenografts in the flank to perform biodistribution experiments as well as 60-min PET scans with small animal PET cameras. The chemical instability of compound 5.8 (Scheme 6, Figure 1) was known at the time

Y₁R Antagonistic PET Ligands 135 of the preparation of the hot analog 5.8-F18, but an alternative PET ligand was not yet available, when this proof-of-concept study was started.

After injection of PET ligand 5.8-F18 most of the radioactivity was detected over the whole scan (60 min) in a part of the intestines, probably the cecum (Figure 3, A + B).

Figure 3. PET images (color table: NIH + white) of a male NMRI (nu/nu) mouse bearing a subcutanous SK-N-MC xenograft in the right flank after intravenous (tail vein) injection of the ¹⁸F-labeled Y₁R antagonist 5.8-F18. The PET scan was started 5 min after injection and images were acquired as six 5-min frames and three 10-5-min frames. Sets of corresponding coronal, sagittal and transaxial sections (planes are indicated by red arrows) are shown for the first (A) and last (B + C) frame of the scan. Already 5 min after injection most radioactivity was detected in a part of the gastrointestinal tract, probably in the cecum, where it was retained for more than 60 min. An accumulation of radioactivity in the brain was clearly detectable in the last frame (55 – 65 min after injection) (C, image depicted with lower threshold).

An accumulation of radioactivity in the SK-N-MC xenograft in the right flank was not observed.

Chapter 5 136

blood

lunge liver pan

creas spleen

intestine kidney

adr. glands musc

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r

% Injected Dose / g

0 2 4 6 8

Figure 4. Biodistribution of 5.8-F18 in NMRI (nu/nu) mice bearing subcutaneous SK-N-MC xenografts 30 min after injection of the PET ligand (n = 3).

Compound 5.9-F18 and potential metabolites, respectively, were excreted either to the gall and urinary bladder as becomes obvious from Figure 5A and Figure 6. In both cases the administration of the PET ligand resulted in a moderate accumulation of radioactivity in the brain (examplary shown for 5.8-F18 in Figure 1C).

Figure 5. PET images (color table: NIH + white) of a male NMRI (nu/nu) mouse bearing a subcutanous SK-N-MC xenograft in the left flank after intravenous (tail vein) injection of the ¹⁸F-labeled Y1R antagonist 5.9-F18. A 60-min PET scan was started 10 min after injection and images were acquired as 5-min frames. A1: Coronal PET image of frame 2 (15 – 20 min after injection). A2: Coronal PET image of frame 12 (65 – 70 min after injection). B1: Coronal PET image of frame 2 at lower threshold; B2: Corresponding transaxial image (planes are indicated by red arrows). The biliary and renal excretion of 5.9-F18 and metabolites, respectively, becomes obvious from images A1 and A2 (highest intensity in the gall and urinary bladder). The SK-N-MC tumor (open arrow) could be visualized only in the first three frames (B1 and B2 show images of frame 2).

Y₁R Antagonistic PET Ligands 137

Figure 6. Biodistribution of 5.9-F18 in NMRI (nu/nu) mice with and without subcutaneous SK-N-MC xenografts 90 min after injection of the PET ligand (n = 4, *n = 2).

Whereas imaging of the Y₁R expressing SK-N-MC tumor was sucessful in the early phase of the PET scan after injection of 5.9-F18 (Figure 5B), the xenograft could not be detected after administration of 5.8-F18.

While biodistribution data correlate well with the PET images in case of the experiments with PET ligand 5.9-F18 (Figure 6), the amount of radioactivity in the intestine after administration of 5.8-F18 appears to be too low compared to the PET images (Figures 3 and 4). Unfortunately, the radioactivity was determined in the whole gastrointestinal (GI) tract and not in individual organ segments. As the activity was normalized by the weight of the sample, potential local enrichment of activity inside the GI tract was not resolved in biodistribution studies with 5.8-F18 (Figure 4).

As already mentioned in the introduction, most PET ligands, which enable tumor imaging, have logP values < 0. Calculated logP values of the presented potential PET ligands are in the range of 1 - 4 indicating a too low water solubility of these compounds (Table 3). 4-Fluorobenzoylation leads to a considerable higher lipophilicity compared to 2-fluoropropionylation as becomes obvious from the logP values computed for compounds which differ only in the fluorinated acyl moiety (5.16 and 5.17 as well as 5.18 and 5.19, Table 3).

Chapter 5 138

Table 3. Structures and computed logP values of BIBP 3226 and the potential PET ligands 5.8 - 5.19 as well as 5.21 for the depicted tautomeric forms.

H(R)

acalculated with ChemDraw Ultra 11.0; ^bcalculated with ACDLABS 9.0; no entry:

calculation was not possible with the respective software

Protonation of the basic guanidine group under physiological conditions leads to an increase in water solubility, but this is insufficient to prevent a biliary excretion of the compounds presented in this chapter. The partial renal excretion of PET ligand 5.9-F18 can be attributed to a partial metabolic conversion of this compound, which should be investigated e.g. by thin layer chromatography analysis of the urine of the mouse in future studies.

As these PET and biodistribution experiments are preliminary studies, co-administration of a non-labeled Y1R antagonist to prevent specific binding of the tracer was not performed. This kind of experiment was envisaged in the case of successful tumor imaging. Therefore, it is not possible to state that the accumulation in the brain (Figure 3C) or the visualization of the tumor (Figure 5B) was Y₁ receptor mediated.

The ¹⁸F-labeled analog of compound 5.17 (Ki = 7 nM) was synthesized as the third PET ligand of this series in the research laboratories of Bayer Schering Pharma AG (Department of Oncology Imaging Research, Berlin) by acylation of amine precursor 4.27 with N-succinimidyl 4-[¹⁸F]fluorobenzoate. Unfortunately, the preparation of the hot analog of the 2-fluoropropionylated

Y₁R Antagonistic PET Ligands 139 high affinity ligand 5.16 (K_i = 1.3 nM) was impossible since 4-nitrophenyl-2-[¹⁸F]fluoropropionate is only routinely used in Munich. PET ligand 5.17-F18 was prepared and administered intravenously to estrogen substituted nude mice with subcutaneous Y₁R-expressing MCF-7-Y₁ tumors (for tumor induction same MCF-7-Y₁ cells as used in previous studies in our laboratory were provided, cf. chapter 2). The experiment revealed a strong hepatobiliary excretion pattern of this compound, and an accumulation in the tumor was not observed (20-min PET scans were started 40 min after injection, n = 3). This can be explained by the unfavorable physicochemical properties of this compound, which is even more lipophilic than the PET ligand 5.9-F18 (ACDLABS 9.0 computed logP values: 2.5 and 4.4, resp.).