68Ga-Labeled DOTA-conjugated sCCK8[Phe2(p-CH2SO3H),Nle3,6], a sulfated cholecystokinin 8 (sCCK8) peptide derivative


Chopra A.

Publication Details



In vitro Rodents



The three subtypes of the cholecystokinin (CCK) receptor (CCKR), designated CCK1R, CCK2R, and CCK2i4svR, belong to the G-protein–coupled receptor family and are classified on the basis of their affinities for the CCK and the gastrin peptides, differential distribution in the tissues, and molecular structure (1). The CCK1R (previously known as CCKA) is found mainly on the peripheral cells of the pancreas and has a 500- to 1,000-fold higher affinity for sulfated CCK8 (sCCK8; contains a sulfated tyrosine (Tyr) residue) compared with the non-sulfated ligand. In addition, CCK1R is expressed mainly in organs of the gastrointestinal tract of rodents, but it is expressed at low levels in humans (2). CCK2R (previously known as CCKB, also designated as the gastrin receptor (GR)) is expressed in the brain, stomach, pancreas, and gallbladder and exhibits an almost equivalent affinity for gastrin and sulfated or non-sulfated CCK. Although CCK1R and CCK2R/GR are expressed in several normal tissues, CCK2R is overexpressed in most cancerous tumors, such as medullary thyroid carcinomas (>90%), astrocytomas (>65%), and stromal ovarian cancers (100%). The CCK2i4svR is a splice variant of the CCK2R that is expressed only in colorectal and pancreatic neoplasms and is not detectable in normal colorectal mucosa (1). Several gastrin and CCK8 analogs that have a high affinity for CCK2R have been developed and labeled with radionuclides for the detection and therapy of these cancers. It has been shown that radiolabeled CCK8 derivatives and minigastrin (MG0; a shorter version of human gastrin 1 that consists of amino acids 5–17 of the parent peptide) can be used with scintigraphy to detect colorectal or pancreatic cancer tumors that overexpress CCK2i4svR (3) or to screen for patients who may benefit from the radiotherapy of this disease (4). In preclinical studies, however, a high uptake of radioactivity in the kidneys was observed with these radiochemicals, and it was concluded that these tracers will be of limited use in humans (5). In addition, the sulfated Tyr residue at position 2 in sCCK8 is rapidly hydrolyzed, and the two methionine (Met) residues at positions 3 and 6 in the peptide can be easily oxidized during radiolabeling procedures or under in vivo conditions (6).

Two cyclized MG analogs, cyclo-MG1 and cyclo-MG2, were synthesized, labeled with 99mTc, and evaluated with single-photon emission computed tomography (SPECT) for the detection of tumors that express CCKR (7). Although the two 99mTc-labeled cyclo-compounds were suitable for the detection of tumors, they had low in vitro stability and generated low-quality scintigraphic images because they rapidly degraded under in vivo conditions. Similar observations have been reported with other 99mTc-labeled compounds that were used in clinical trials for the visualization of tumors that overexpressed the CCK2R (8). In an ongoing effort to develop imaging agents that can be used to visualize tumors that express CCK2R, a stabilized sCCK8 derivative was prepared by replacing the sulfated Tyr in the molecule with a sulfated phenylalanine (Phe2(p-CH2SO3H)) and by substituting the Met residues in the peptide with either norleucine (Nle) (sCCK8[Phe2(p-CH2SO3H),Nle3,6]) or homoproparglyglycine (HPG; sCCK8[Phe2(p-CH2SO3H),HPG3,6]) (6). To facilitate the radiolabeling of these compounds with radionuclides such as 111In or 68Ga, the different peptides, including sCCK8, were conjugated with 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA). In one study the sCCK8, sCCK8[Phe2(p-CH2SO3H), sCCK8[Phe2(p-CH2SO3H),Nle3,6] and sCCK8[Phe2(p-CH2SO3H),HPG3,6] peptides were labeled with 111In, and their biodistribution was compared in athymic BALB/c nude mice bearing AR42J cell tumors that express CCK2R (6). In another study, the biodistribution and noninvasive imaging characteristics of [111In/68Ga]-DOTA-sCCK8[Phe2(p-CH2SO3H),Nle3,6] were compared with those of [111In/68Ga]-DOTA-sCCK8 and [111In/68Ga]-DOTA-MG0 in athymic BALB/c nude mice bearing tumors generated from A431 cells transfected with CCK2R (A431-CCK2R cells) (9). This chapter describes studies performed only with the 68Ga-labeled peptides. Results obtained with the 111In-labeled peptides are described in a separate chapter of MICAD (www.micad.nih.gov) (10).



The synthesis of [68Ga]-DOTA-sCCK8, [68Ga]-DOTA-sCCK8[Phe2(p-CH2SO3H),Nle3,6], and [68Ga]-DOTA-MG0 has been described elsewhere (9). The radiochemical yield, radiochemical purity, and specific activity of the 68Ga-labeled peptides were not reported (9).

The 68Ga-labeled peptides used for positron emission tomography (PET) imaging were reported to have a specific activity of 50–58 MBq/nmol (1.85–2.15 mCi/nmol).

In Vitro Studies: Testing in Cells and Tissues


A competitive binding assay with A431-CCK2R cells was used to determine the apparent IC50 values of nonradioactive 69Ga-conjugated DOTA-sCCK8, DOTA-sCCK8[Phe2(p-CH2SO3H),Nle3,6], and DOTA-MG0 (9). The IC50 values for the peptides were reported to be 0.83 ± 0.08 nM, 16.54 ± 0.06 nM, and 5.91 ± 0.06 nM, respectively. This indicated that, among the different CCK2R ligands used in this study, the [69Ga]-DOTA-sCCK8[Phe2(p-CH2SO3H),Nle3,6] peptide had the lowest affinity for the receptor.

Animal Studies



The biodistribution of [68Ga]-DOTA-sCCK8, [68Ga]-DOTA-sCCK8[Phe2(p-CH2SO3H),Nle3,6], and [68Ga]-DOTA-MG0 was investigated in athymic BALB/c nude mice bearing A431-CCK2R cell tumors (9). The animals (n = 5 mice/group for each labeled peptide) were injected intravenously with 2 MBq (74 μCi; 0.03 nmol) of the peptide, and the rodents were euthanized at 1 h postinjection (p.i.) to determine the amount of radioactivity that accumulated in the various tissues, including the tumors. For blocking studies, the animals (n = 3 mice/labeled peptide) were injected with the tracer in the presence of 1,000-fold molar excess nonradioactive DOTA-sCCK8. Results obtained from this study were presented as percentage of injected dose per gram tissue (% ID/g).

With [68Ga]-DOTA-sCCK8 and [68Ga]-DOTA-sCCK8[Phe2(p-CH2SO3H),Nle3,6], the uptake of radioactivity in the tumors was 7.48 ± 0.42% ID/g and 6.35 ± 1.41% ID/g, respectively, compared with 14.2 ± 4.06% ID/g for [68Ga]-DOTA-MG0. The uptake of label in the pancreas was highest with [68Ga]-DOTA-sCCK8 (~13.0% ID/g), followed by [68Ga]-DOTA-MG0 (~6.0% ID/g) and [68Ga]-DOTA-sCCK8[Phe2(p-CH2SO3H),Nle3,6] (~2.5% ID/g) (9). A very high accumulation of tracer was observed in the kidneys (>75% ID/g) with [68Ga]-DOTA-MG0, compared with <1.0% ID/g for the other two labeled peptides. All other organs (blood, lungs, muscle, spleen, and small intestines) showed label accumulation of <0.5% ID/g with each of the 68Ga-labeled peptides. Accumulation of tracer in the different tissues was blocked by at least 50% when an excess concentration of nonradioactive sCCK8 was co-injected with the radiolabeled peptides. This indicated that the uptake of label in the tumors and the other tissues was mediated specifically through CCK2R.

MicroPET/computed tomography images of mice injected with the different 68Ga-labeled peptides were acquired for 30 min as described by Joosten et al. (9). The tumors were clearly visible at 1 h p.i. with each of the labeled peptides. In addition to the tumors, the intestines of the animals were visible with both [68Ga]-DOTA-sCCK8 and [68Ga]-DOTA-sCCK8[Phe2(p-CH2SO3H),Nle3,6]. Radioactivity was also detected in the bladder with [68Ga]-DOTA-sCCK8[Phe2(p-CH2SO3H),Nle3,6]. With [68Ga]-DOTA-MG0, a very high uptake of label was observed in the kidneys, but very little radioactivity was detected in these organs with the other two labeled peptides.

From these studies, the investigators concluded that the 68Ga-labeled analogs of sCCK8 are probably suitable for the noninvasive visualization and characterization of CCK2R in rodents. However, more work is necessary to exploit the full potential of these probes for the PET imaging of CCKR (9).

Other Non-Primate Mammals


No reference is currently available.

Non-Human Primates


No reference is currently available.

Human Studies


No reference is currently available.

Supplemental Information


No information is currently available.


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