Development of [¹²³I]IPEB and [¹²³I]IMPEB as SPECT Radioligands for Metabotropic Glutamate Receptor Subtype 5

mGlu₅ play an important role in physiology and pathology to various central nervous system (CNS) diseases. Several positron emission tomography (PET) radiotracers have been developed to explore the role of mGlu₅ in brain disorders. However, there are no single photon emission computed tomography (SPECT) radioligands for mGlu₅. Here we report development of [¹²³I]­IPEB ([¹²³I]<b>1</b>) and [¹²³I]­IMPEB ([¹²³I]<b>2</b>) as mGlu₅ radioligands for SPECT. [¹²³I]<b>1</b> and [¹²³I]<b>2</b> were produced by copper­(I) mediated aromatic halide displacement reactions. The SPECT imaging using mouse models demonstrated that [¹²³I]<b>1</b> readily entered the brain and accumulated specifically in mGlu₅-rich regions of the brain such as striatum and hippocampus. However, in comparison to the corresponding PET tracer [¹⁸F]­FPEB, [¹²³I]<b>1</b> showed faster washout from the brain. The binding ratios of the striatum and the hippocampus compared to the cerebellum for [¹²³I]<b>1</b> and [¹⁸F]­FPEB were similar despite unfavorable pharmacokinetics of [¹²³I]<b>1</b>. Further structural optimization of <b>1</b> may lead to more viable SPECT radiotracers for the imaging of mGlu₅

Summary of PEG-like Nanoprobes (PN’s).

<p>*M.W. Obs. = molecular weights were determined from mass spectrometry results.</p><p>**Volumes are expressed as diameters in nm (to enable comparison with nanomaterials) and as the equivalent volumes of proteins (to enable comparison with proteins).</p><p>***Values are means±1 S.D.</p

Role PEG in determining elimination by surface fluorescence imaging.

<p><b>A</b>) Animals were injected with unPEGylated peptide and imaged at the times indicated. Bar graph gives whole animal surface fluorescence determined as means and standard deviations, n = 6. <b>B</b>) With the 5 kDa PEG attached, PN(783)4.3 is obtained, which shows renal elimination within 20 minutes (arrow). <b>C</b>) With the 30 kDa PEG, PN(783)10.0 is obtained, which shows a long vascular phase followed by elimination. Biodistribution and elimination of ¹¹¹In-PN(783)10.0 is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095406#pone-0095406-g006" target="_blank">Figure 6</a>.</p

Multimodal imaging of EPR tumor targeting and elimination of PN(783)10.0.

<p><b>A</b>) SPECT/CT images of two mice bearing two HT-29 tumors as a function of time after injection. At 2 h post injection, agent is in the blood and interstitium. By 24 h post-injection tumors are becoming apparent as agent is being cleared. At 48 h, labeling is highly tumor selective. <b>B</b>) Surface fluorescence imaging of two additional mice bearing the same tumor. By surface fluorescence, as with SPECT, labeling is highly tumor selective at 48 h. <b>C</b>) Organ biodistribution was obtained by dissection and ¹¹¹In counting at 24 h and 48 h post-injection. Data are means and standard deviations, n = 5. <b>D</b>) A whole animal radioactivity elimination curve. Data are means and standard deviations, with extremely small standard deviations, n = 5.</p

Variable PN pharmacokinetics analyzed by the two compartment pharmacokinetic model in normal mice.

<p><b>A</b>) Two compartment pharmacokinetic model showing three microscopic rate constants. Serum fluorescence for PN(783)10.0 <b>B</b>) and PN(783)4.3 <b>C</b>) after injection are shown. Data were fit to the two compartment model with data provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095406#pone-0095406-t001" target="_blank">Table 1</a>. <b>D)</b> Time courses for blood and interstitial fluorescence of PN(783)10.0 using microkinetic constants from <b>B</b>).</p

Fluorescent imaging of three pharmacokinetic phases of PN’s with diameters of 10 nm.

<p><b>A</b>) Vascular phase, two photon microscopy of brain vasculature of normal mice. Mice underwent a craniotomy and implantation of a transparent window. Vessel intensity drops due vascular escape, but there is no interstitial fluorescence in the brain due to the blood brain barrier. Scale marker = 50 microns. <b>B</b>) Intravital confocal microscopy of the vascular and interstitial phases of an mCherry expressing HT-29 xenograft. During the vascular phase (10 min post-injection), vessels are imaged, without interstitial fluorescence. During the interstitial phase (20 h post-injection), interstitial fluorescence is prominent. Scale marker = 20 microns. <b>C</b>) Surface fluorescence/X-ray imaging of the tumor retention phase of PN(545)10.0. Shown are the HT-29/mCherry tumor with the skin removed as a white light image, mCherry tumor fluorescence (green), PN(545)10.0 fluorescence (purple) and the green/purple over lay (white). <b>D</b>) Confocal microscopy of the tumor retention phase of PN(497)10.0. Shown are a sectioned HT-29 mCherry expressing tumor with nuclei stained blue (DAPI), mCherry tumor cells (red), PN(497)10.0 (green) and a green/red overlay (yellow).</p

Reaction with PEG polymers and fluorochrome and DOTA bearing peptides yields PEG-like Nanoprobes (PN’s).

<p><b>A</b>) Syntheses of PEG-like Nanoprobe (PN’s). The peptide (DOTA)Lys-Cys(Fluor.), with an N-terminal DOTA, a variable fluorochrome (Fluor.) attached to the cysteine side chain, and a single primary amine, reacts with the NHS ester of a variable PEG. In the conventional PEGylations of proteins or nanoparticles, multiple PEG’s provide a PEG bearing surface. This approach yields materials with a PEG to protein ratio or with a PEG surface density. <b>B</b>) Synthesis of the PN denoted PN(783)4.3. A 5 kDa PEG is reacted with the (DOTA)Lys-Cys(IR-783) peptide. The diameter (by FPLC) is 4.3 nm and the absorption maximum is 783 nm, hence PN(783)4.3. <b>C</b>) A 30 kDa PEG is reacted with the (DOTA)Lys-Cys(Cy3) peptide. The diameter is 10.0 nm and an absorption maximum is 545 nm, hence, PN(545)10.0. A list of PN’s and their properties is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095406#pone-0095406-t001" target="_blank">Table 1</a>.</p

Stability of PN’s in mouse serum.

<p>The stability of PN(783)4.3 <b>A</b>) or PN(783)10.0 <b>B</b>) was examined by incubating nanoprobes for the indicated times and subjecting samples to FPLC. Arrows are the retention times of PN’s from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095406#pone-0095406-g002" target="_blank"><b>Figure 2A</b></a>, color coded as in that figure. Fluorochrome size is unchanged for both PN’s. <b>C</b>) Stability of PN fluorescence. PN(783)4.3 or PN(783)10.0 were incubated as indicated and fluorescence determined. <b>D</b>) Stability of ¹¹¹In binding to PN’s. ¹¹¹In-PN(783)4.3 or ¹¹¹In-PN(783)10.0 were incubated as indicated and radioactivity associated with the PN determined by HPLC. Exemplary chromatograms are provided; see <b>Figure S6</b> in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095406#pone.0095406.s001" target="_blank">File S1</a>.</p