Bioconjugate Chem. 2007, 18, 1516−1520
1516
Radiolabeling of Bombesin-Like Peptide with Biodistribution in Rats†
99mTc: 99mTc-Litorin
and
Kubra Durkan, Fatma Yurt Lambrecht,* and Perihan Unak Department of Nuclear Applications, Institute of Nuclear Sciences, Ege University, Bornova 35100, Izmir, Turkey. Received December 26, 2006; Revised Manuscript Received July 19, 2007
Bombesin-like peptides are related to several human cancer receptors, including small cell lung, prostate, breast, colon, and pancreatic cancers. Litorin, an amphibian bombesin peptide derivative, is found to stimulate the contraction of smooth muscle, to stimulate gastrin, gastric acid, and pancreatic secretion, and to suppress the nutriment in in vivo experiments. In the present study, litorin was labeled with 99mTc by the stannous chloride procedure. Labeling yield is 95 ( 1.4%, as determined by radio thin layer chromatography (RTLC) and radio high performance chromatography (RHPLC). Results of in vitro studies demonstrated a high stability in serum and cysteine solutions. In vivo biodistribution was investigated with normal male Albino Wistar rats. Biodistribution data showed fast clearance, low intestinal accumulation, and significant uptake in bombesin/gastrin releasing peptide (BN/GRP) receptor rich tissues such as the pancreas (23.56 ( 0.01 %ID/g 30 min pi). It can be blocked partially by previous administration of ‘cold’ litorin. The results showed specificity of the uptake. As 99mTclitorin displays good radiolabeling and biodistribution, it is a potentially useful radiopharmaceutical for detection of bombesin receptor-expressing cancers.
INTRODUCTION Radiolabeled peptides have exhibited great potential for imaging and therapy of tumors in recent years. Peptides have some favorable characteristics: they are readily synthesized, are inexpensive, and can withstand rigid chemical conditions for modifications and radiolabeling. Furthermore, because of their low molecular weight, small peptides are expected to efficiently penetrate into tumor tissue, exhibit a faster clearance, and thus lead to better tumor-to-background ratios than high molecular weight compounds such as monoclonal antibodies and their constructs (1-7). The amphibian 14-amino acid peptide bombesin (BN) is part of the super family of bombesin-like peptides that also includes the mammalian homologue gastrin relasing peptide (GRP) and the related neuromedin B (NMB) (1). The gastrin releasing peptide has been found to produce a wide range of biological responses in peripheral tissues as well as in the central nervous system, for instance stimulation of gastrointestinal hormone release, exocrine secretion, and maintenance of circadian rhythms (1, 2, 8-11). Bombesin-like peptides exert their effects on target cells by binding to surface G protein-coupled receptors characterized by the typical configuration of seven transmembrane domains (12). They have a wide range of physiological activities, including stimulation of the release of numerous gastrointestinal hormones, stimulation of pancreatic enzyme secretion effects on the central nervous system, such as thermoregulation, and inhibition of thyroid-stimulating hormone (13). In addition to these physiological effects, it also plays an important role in cancer. Initiation of its interaction with the GRP receptor * To whom correspondence should be addressed. Fax: 90 2323886466; e-mail:
[email protected]. † A part of radiolabeling of this study was presented at a poster session of the Technetium, Rhenium and Other Metals in Chemistry and Nuclear Medicine 7, 7th International Symposium on Technetium in Chemistry and Nuclear Medicine, 329-330, Bressanone (BZ), Italy, September 6-9, 2006.
promotes tumor growth in a number of normal and human cancer cells both in culture and in nude mice xenografts (12, 14). On the other hand, it has been established that BN/GRP receptors are expressed in resected human biopsy specimens of mainly small lung cancers, prostate carcinomas, gastric, pancreatic, colon cancer, and breast carcinomas often at much higher densities than in normal surrounding tissue; this provides at molecular basis for GRP-R-targeted diagnostic imaging and/ or internal radiotherapy of malignant lesions with radiolabeled bombesin analogues. In addition, the high frequency of these tumors has had a synergic effect in stimulating research on novel bombesin-like radiotracers for effective in vivo targeting of GRP-R-positive neoplastic disease (12-15). Furthermore, the use of BN/GRP antagonists as carrier molecules for targeting cytotoxic drugs to tumor cells has recently been proposed (12). Some peptides structurally related to bombesin were discovered from amphibian skin and divided into three groups: (1) bombesin family that includes bombesin and alytesin; (2) ranatensin family that includes ranatensin, litorin; (3) their derivatives and the phyllolitorin family (16). Litorin (pGlu-GlnTrp-Ala-Val-Gly-His-Phe-Met-NH2) has an amino acid sequence similar to that of bombesin. Rapid pharmacokinetics is ideal for labeling peptides with a radioisotope that has a short half-life, such as technetium-99m. Among all radioisotopes used in nuclear medicine, Tc-99m is still the most widely applied for diagnostic purposes, mainly because of ready availability, low cost, excellent imaging properties, favorable dosimetry, and high specific activity. The development of 99mTc-labeled BN/GRP analogues for cancer imaging would be even more promising. For this reason, various 99mTc- or 111In-labeled bombesin analogues could probably allow early noninvasive diagnosis of GRP receptorpositive tumors by SPECT in nuclear medicine (1, 2, 8, 1015, 17-23). The aim of the present work is to develop a 99mTclabeled bombesin-like peptide for tumor imaging. Litorin was labeled with Tc-99m, and its radiopharmaceutical potential was investigated with biodistribution studies in normal male Albino Wistar rats.
10.1021/bc060400x CCC: $37.00 © 2007 American Chemical Society Published on Web 08/31/2007
Radiolabeling of Bombesin-Like Peptide with
99mTc
Bioconjugate Chem., Vol. 18, No. 5, 2007 1517
Figure 1. RHPLC chromatograms from radioactivity detection.
EXPERIMENTAL PROCEDURES Materials. Na99mTcO4 was supplied by the Department of Nuclear Medicine of Ege University, as 99Mo/99mTc generator eluent (Monrol, Istanbul-Turkey). Litorin was obtained from Phoenix Pharmaceuticals Inc., Germany. All other chemicals were from Merck Chemical Co. Radiolabeling with 99mTc. Radiolabeling of litorin with Tc99m was performed via the stannous (II) chloride method. Litorin was dissolved in distilled water at 1 mg/50 mL concentration. The solution of peptide was distributed in 10 µg/ 500 µL aliquots in eppendorf vials that were stored at -20 °C. SnCI2·2H2O (1 mg) was freshly prepared by dissolving in 1 mL distilled water and 25 µL of stannous chloride solution was added to the eppendorf vial containing 5 µg/250 µL litorin solutions. pH was adjusted to 3 by using HCI (0.1N) or NH3 (0.1N). After the SnCI2·H2O solution was added to the vial, about 55.5 MBq Na99mTcO4 was added. It was left to incubate for 25 min at room temperature. Radiochemical Analysis. Effects of pH, amount of stannous chloride on labeling percentage, and serum stability time were investigated. Radio-TLC Analysis. RTLC was done with a Sigma ITLC chamber using 10 × 1.5 cm2 RTLC-SG strips. After development, they were dried, covered by sellotape, and cut into 0.5 cm strips. These were counted with a Cd(Te) detector equipped with a RAD 501 single channel analyzer (Isin Electronic Co., Izmir, Turkey). Two different solution systems were used as a developing solution: ACD [citrate-dextrose buffer solution (Eczacibasi-Baxter)] and acetone. Radio-HPLC Analysis. HPLC analysis was performed on a low-pressure gradient HPLC system with LC-10 ATvp quaternary pump, UV detector (Shimadzu SPD-10ATvp, MachereyNagel, EC 250/4.6 Nucleodur 100-5 C18 column) and 20 µL loop and equipped with a Cd(Te) detector equipped with a RAD501 single channel analyzer. HPLC was run using a gradient of 0.1% TFA in H2O (solvent A) and 0.1% TFA in CH3CN (solvent B) at a flow rate of 1 mL/min. The HPLC gradient system was begun with a solvent composition of 95% A and 5% B and reached to 60% A and 40% B in 20 min. The UV detector was settled at 240 and 280 nm. Serum Stability of 99mTc-Litorin. 99mTc-Litorin (300 µL) was incubated with fresh human serum (600 µL) in duplicate at 37 °C through 24 h. During incubation, the sample was
analyzed at 15 min, 1, 2, 3, 4, and 24 h. The amount of free pertechnetate in the samples was determined by RTLC using ACD and acetone as medium. Partition Coefficient. A 100 µL aliquot of radiolabeled peptide was added to a test tube containing 3 mL of each of n-octanol and water. The tube was vortexed, for 1 h at room temperature, and then centrifuged at 3000 rpm for 5 min. Aliquots (0.5 mL) of each phase were taken for counting. The partition coefficient was determined by the function: Partition coefficient ) log10(counts in n-octanol layer/ counts in aqueous layer) (15). Cysteine Challenge. 99mTc-Litorin was tested for instability toward cysteine. A fresh cysteine solution was prepared (0.5 mg mL-1 in 0.1 M PBS, pH 7.0) and diluted to different concentrations. A known amount of the radiolabeled peptides was incubated with an excess amount of cysteine (100 fold molar excess compared to the peptide) at 37 °C for 1 h. Radiochemical purity was determined by RTLC. Biodistribution in Normal Rats. The institutional Animal Review Committee of Ege University approved the animal experiments. Male, 24 weeks rat Albino Wistar rats (average weight 150-200 g) were used for the biodistribution in the groups of three per time point. The pH of 99mTc-litorin (0.1 µg litorin, specific activity 401 GBq/µmol) was adjusted to 7, using 0.1 N NH3. The solution was then passed through a 0.22 µm Millipore filter. 99mTc-Litorin was injected into the tail vein. Animals were sacrificed under intense ether atmosphere at 30, 90, 180, and 300 min postinjection and main organs and blood were removed, weighed, and counted. Biodistribution data were calculated as the percent injected dose per gram tissue (%ID/ g). In ViVo Blocked Experiments. Receptor blocking studies were also carried out where cold litorin was administered to the animals for in ViVo saturation of GRP receptors prior to an injection of 99mTc-litorin. In this study, each animal received 1 µg of cold litorin i.v., 30 min prior to the injection of 99mTclitorin (specific activity 401 GBq/µmol). They were sacrificed at 30 min postinjection, the main tissues were removed, the radioactivity was counted, and %ID/g tissue was calculated. Statistical Analysis. Differences in the mean values of measured activities were evaluated statistically by SPSS 10 program (Univariate Variance Analyses and Pearson Correlation). Probability values
