Department of Nuclear Medicine, Radboud University Nijmegen Medical Center, The Netherlands.
Bioconjug Chem. 2010 Apr 21;21(4):663-70. doi: 10.1021/bc900465y.
Radiolabeled cholecystokinin-8 (CCK8) peptide analogues can be used for peptide receptor radionuclide imaging and therapy for tumors expressing CCK2/gastrin receptors. Earlier findings indicated that sulfated CCK8 (sCCK8, Asp-Tyr(OSO(3)H)-Met-Gly-Trp-Met-Asp-Phe-NH(2)) may have better characteristics for peptide receptor radionuclide therapy (PRRT) than gastrin analogues. However, sCCK8 contains an easily hydrolyzable sulfated tyrosine residue and two methionine residues which are prone to oxidation. Here, we describe the synthesis of stabilized sCCK8 analogues, resistant to hydrolysis and oxidation. Hydrolytic stability was achieved by replacement of the Tyr(OSO(3)H) moiety by a robust isosteric sulfonate, Phe(p-CH(2)SO(3)H). Replacement of methionine by norleucine (Nle) or homopropargylglycine (HPG) avoided undesired oxidation side-reactions. The phenylalanine analogue Phe(p-CH(2)SO(3)H) of l-tyrosine, synthesized by a modification of known synthetic routes, was incorporated in three peptides: sCCK8[Phe(2)(p-CH(2)SO(3)H),Met(3,6)], sCCK8[Phe(2)(p-CH(2)SO(3)H),Nle(3,6)], and sCCK8[Phe(2)(p-CH(2)SO(3)H),HPG(3,6)]. All peptides were N-terminally conjugated with the macrocyclic chelator DOTA (1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid) and radiolabeled with In-111. In vitro binding assays on CCK2R-expressing HEK293 cells revealed that all three peptides showed specific binding and receptor-mediated internalization, with binding affinity values (IC(50)) in the nanomolar range. In vitro oxidation studies demonstrated that peptides with Nle or HPG indeed were resistant to oxidation. In vivo targeting studies in mice with AR42J tumors showed that tumor uptake was highest for (111)In-DOTA-sCCK8 and (111)In-DOTA-sCCK8[Phe(2)(p-CH(2)SO(3)H),Nle(3,6)] (4.78 +/- 0.64 and 4.54 +/- 1.15%ID/g, respectively, 2 h p.i.). The peptide with the methionine residues replaced by norleucine ((111)In-DOTA-sCCK8[Phe(2)(p-CH(2)SO(3)H), Nle(3,6)]) showed promising in vivo characteristics and will be further investigated for radionuclide imaging and therapy of CCK2R-expressing tumors.
放射性标记胆囊收缩素-8（CCK8）肽类似物可用于表达 CCK2/胃泌素受体的肿瘤的肽受体放射性核素成像和治疗。早期研究结果表明，磺化 CCK8（sCCK8，Asp-Tyr（OSO3H）-Met-Gly-Trp-Met-Asp-Phe-NH2）可能比胃泌素类似物具有更好的肽受体放射性核素治疗（PRRT）特性。然而，sCCK8 含有一个易于水解的磺化酪氨酸残基和两个易氧化的蛋氨酸残基。在这里，我们描述了合成稳定的 sCCK8 类似物的方法，使其具有抗水解和氧化的特性。通过用稳定的磺酸酯取代 Tyr（OSO3H）部分来实现水解稳定性，得到苯丙氨酸类似物 Phe（p-CH2SO3H）。用正亮氨酸（Nle）或高丙氨酸（HPG）取代蛋氨酸可以避免不必要的氧化副反应。通过对已知合成路线的修改，合成了 l-酪氨酸的苯丙氨酸类似物 Phe（p-CH2SO3H），并将其引入到三种肽中：sCCK8[Phe（2）（p-CH2SO3H），Met（3，6）]，sCCK8[Phe（2）（p-CH2SO3H），Nle（3，6）]和 sCCK8[Phe（2）（p-CH2SO3H），HPG（3，6）]。所有的肽都在 N 端与大环螯合剂 DOTA（1,4,7,10-四氮杂环十二烷-N,N',N'',N''-四乙酸）连接，并与 In-111 进行放射性标记。在表达 CCK2R 的 HEK293 细胞上进行的体外结合实验表明，所有三种肽均表现出特异性结合和受体介导的内化，其结合亲和力值（IC50）均在纳摩尔范围内。体外氧化研究表明，含有 Nle 或 HPG 的肽确实具有抗氧化能力。在 AR42J 肿瘤的小鼠体内靶向研究中，（111）In-DOTA-sCCK8 和（111）In-DOTA-sCCK8[Phe（2）（p-CH2SO3H），Nle（3，6）]的肿瘤摄取量最高（分别为 4.78±0.64 和 4.54±1.15%ID/g，2 h p.i.）。用正亮氨酸取代蛋氨酸的肽（（111）In-DOTA-sCCK8[Phe（2）（p-CH2SO3H），Nle（3，6）]）表现出有希望的体内特征，将进一步研究其用于 CCK2R 表达肿瘤的放射性核素成像和治疗。
