Receptor-targeted optical imaging of tumors with near-infrared fluorescent ligands.

We report here the in vivo diagnostic use of a peptide-dye conjugate consisting of a cyanine dye and the somatostatin analog octreotate as a contrast agent for optical tumor imaging. When used in whole-body in vivo imaging of mouse xenografts, indotricarbocyanine-octreotate accumulated in tumor tissue. Tumor fluorescence rapidly increased and was more than threefold higher than that of normal tissue from 3 to 24 h after application. The targeting conjugate was also specifically internalized by primary human neuroendocrine tumor cells. This imaging approach, combining the specificity of ligand/receptor interaction with near-infrared fluorescence detection, may be applied in various other fields of cancer diagnosis.

Synthesis, characterization, and biological properties of cyanine-labeled somatostatin analogues as receptor-targeted fluorescent probes.

We present the synthesis and characterization of the somatostatin receptor-specific peptide H(2)N-(D-Phe)-cyclo[Cys-Phe-(D-Trp)-Lys-Thr-Cys]-Thr-OH, which is labeled with a carboxylated indodicarbo- and an indotricarbocyanine dye at the N-terminal amino group. The preparation was performed by automated solid-phase synthesis, with subsequent attachment of the cyanine dye and cleavage of the entire conjugate from the resin. The compounds display high molar absorbance and fluorescence quantum yields typical for cyanine dyes and are thus suitable receptor-targeted contrast agents for molecular optical imaging. The ability of these agents to target the somatostatin receptor was demonstrated by flow cytometry in vitro, in which the indotricarbocyanine conjugate led to elevated cell-associated fluorescence on somatostatin receptor-expressing tumor cells. In contrast, the corresponding linearized derivative of the sequence H(2)N-(D-Phe)-Met-Phe-(D-Trp)-Lys-Thr-Met-Thr-OH produced only minimal cell fluorescence, hence confirming the specificity of the cyclic somatostatin analogue. Intracellular localization could be visualized by near-infrared (NIR) fluorescence microscopy. In conclusion, receptor-specific peptides are promising tools for designing site-directed optical contrast agents for use in molecular optical imaging.

Vasoactive intestinal peptide receptor scintigraphy in patients with pancreatic adenocarcinomas or neuroendocrine tumours.

Human adenocarcinomas of the gastroenteropancreatic system overexpress vasoactive intestinal peptide (VIP) receptors and therefore represent logical diagnostic targets for receptor scintigraphy. Using iodine-123 labelled VIP, the newly employed diagnostic procedure termed VIP receptor scintigraphy (VIP-RS) appears to detect tumour tissue, especially pancreatic metastatic tumours, in almost all cases. So far, however, only a single centre has demonstrated convincing positive results. The aim of this study was to compare the sensitivity and specificity of VIP-RS with those of computer tomography (CT) and transabdominal ultrasound in patients with extensive pancreatic metastatic adenocarcinomas and neuroendocrine tumours. VIP was radiolabelled with carrier-free 123I using the chloramine T-method and preparative high-performance liquid chromatography for purification. Patients with metastatic pancreatic (n=12) and colorectal (n=3) carcinomas (adenocarcinoma: n=13, neuroendocrine tumour: n=2) were studied by VIP-RS, CT, ultrasound and, in one case, also by radioligand receptor autoradiography. Carrier-free radioiodinated VIP of maximum specific radioactivity maintained a high biological activity as determined by cAMP formation in receptor-expressing tumour cell lines. Intravenous injection of 123I-VIP did not cause any side-effects. Biodistribution, determined over 24 h, was high in the lungs and low in abdominal organs. Although all patients had extensive metastatic disease as evidenced by CT and ultrasound, VIP-RS was unable to detect either primaries or metastases in these patients. Only in two patients could a significant uptake of radiolabel be detected in organs directly infiltrated by the primary. To exclude false-negative findings, tumour tissue in one patient with a large primary, undetectable by VIP-RS, was analysed by radioligand receptor autoradiography and shown to be receptor positive. Moreover, in vitro receptor determinations showed that pancreatic carcinomas usually have fewer VIP receptors than the normal tissues to which they metastasize, like the liver. It is concluded that VIP can be radioactively labelled with maximum specific radioactivity while maintaining biological activity. Intravenous administration leads to a biodistribution almost identical to that reported previously. However, in contrast to these reports, very low sensitivity and specificity were observed for the detection of pancreatic cancers. In retrospect, these findings are not surprising since VIP receptor expression was observed to be higher in normal tissues than in neoplastic ones.

Evidence for circadian variations of thyroid hormone concentrations and type II 5'-iodothyronine deiodinase activity in the rat central nervous system.

The 24-h patterns of tissue thyroid hormone concentrations and type II 5'- and type III 5-iodothyronine deiodinase (5'D-II and 5D-III, respectively) activities were determined at 4-h intervals in different brain regions of male euthyroid rats entrained to a regular 12-h light/12-h dark cycle (lights on at 6:00 a.m.). Activity of 5'D-II, which catalyzes the intracellular conversion of thyroxine (T4) to 3,3',5-triiodo-L-thyronine (T3) in the CNS, and the tissue concentrations of both T4 and T3 exhibited significant daily variations in all brain regions examined. Periodic regression analysis revealed significant circadian rhythms with amplitudes ranging from 9 to 23% (for T3) and from 15 to 40% (for T4 and 5'D-II) of the daily mean value. 5'D-II activity showed a marked nocturnal increase (1.3-2.1-fold vs. daytime basal value), with a maximum at the end of the dark period and a minimum between noon and 4:00 p.m. 5D-III did not exhibit circadian patterns of variation in any of the brain tissues investigated. Our results disclose circadian rhythms of 5'D-II activity and thyroid hormone concentrations in discrete brain regions of rats entrained to a regular 12:12-h light-dark cycle and reveal that, in the rat CNS, T3 biosynthesis is activated during the dark phase of the photoperiod. For all parameters under investigation, the patterns of variation observed were in part regionally specific, indicating that different regulatory mechanisms may be involved in generating the observed rhythms.

Effects of lithium and carbamazepine on thyroid hormone metabolism in rat brain.

The effects of lithium (LI) and carbamazepine (CBM) on thyroid hormone metabolism were investigated in 11 regions of the brain and three peripheral tissues in rats decapitated at three different times of day (4:00 A.M., 1:00 P.M., and 8:00 P.M.). Interest was focused on the changes in the two enzymes that catalyze: (1) the 5'deiodination of T4 to the biologically active T3, i.e., type II 5'deiodinase (5'D-II) and (2) the 5 (or inner-ring) deiodination of T3 to the biologically inactive 3'3-T2, i.e., type III 5 deiodinase (5D-III). A 14-day treatment with both LI and CBM induced significant reductions in 5D-III activity. However, 5'D-II activity was elevated by CBM and reduced by LI, both administered in concentrations leading to serum levels comparable with those seen in the prophylactic treatment of affective disorders. The effects were dose dependent, varied according to the region of the brain under investigation, and strongly depended on the time of death within the 24-hour rhythm. The consequences of these complex effects of LI and CBM on deiodinase activities for thyroid hormone function in the CNS and also their possible involvement in the mechanisms underlying the mood-stabilizing effects of both LI and CBM remain to be investigated.

Carbamazepine affects triiodothyronine production and metabolization in rat hippocampus.

The effects of subchronic administration of carbamazepine on thyroid hormone metabolism were investigated in the hippocampus in adult male rats at two different measuring times (4 a.m. and 8 p.m.). Carbamazepine enhanced the activity of 5'II-deiodinase, which catalyzes the deiodination of the prohormone T4 to the active compound T3, at 8 p.m., but not at 4 a.m. The activity of 5III-deiodinase, which catalyzes the further deiodination of the active hormone T3 to its metabolite 3,3'T2, was inhibited at 4 a.m. but not at 8 p.m. These effects of carbamazepine on intracellular thyroid hormone metabolism in the hippocampus should theoretically lead to a rise in T3 production. It remains to be investigated whether they are somehow involved in the as yet unknown mechanisms underlying the anticonvulsant/mood-stabilizing effects of carbamazepine.