Labeling and distribution of linear peptides identified using in vivo phage display selection for tumors

To develop targeting molecules to be used for vascular targeting of short half-lived -emitters for radioimmunotherapy, linear peptide phage display libraries were selected in vivo for binding to IC-12 rat tracheal tumors growing in severe combined immune deficient mice. After three rounds of selection, 15 phage clones were analyzed for DNA sequence, and the deduced translation products of cDNA inserts were compared. Three consensus sequences were chosen from three separate experimental selection series and peptides of these sequences with added -gly-gly-tyr were obtained. Peptides were radiolabeled on tyrosine with ¹²⁵I and the biodistribution in tumor-bearing mice was determined. The radioiodinated peptides were stable in vitro and when injected in tumor-bearing mice 3.0 %ID/g accumulated in the tumor; however, much of the ¹²⁵I was found in the gastrointestinal tract and thyroid, indicative of dehalogenation of the labeled peptide. Radiolabeling peptide 2 with N-succinimidyl-3-¹²⁵I-iodobenzoate resulted in faster excretion, which in turn resulted in lower levels in tumor and other organs, especially thyroid and gastrointestinal tract. Peptide 2 was derivatized with the bifunctional isothiocyanates of cyclohexyl-B diethylenetriaminepentaacetic acid (DTPA) or CHX-A″ DTPA by direct conjugation or with a hydroxylamine derivative of 1B4M-DTPA (2-(p-[O-(carboxamylmethyl)hydroxylamine]benzyl)-6-methyl-diethylenetriamine-N,N,N′,N″,N″-pentaacetic acid ) coupled at the N-terminus. The primary molecular species in the conjugated products were shown by mass spectrometry to have one DTPA per peptide. Peptide chelate conjugates were radiolabeled with ²¹³Bi and the products tested for biodistribution in tumor-bearing mice. The data show that chelation of ²¹³Bi to peptides was accomplished by both the direct method of DTPA attachment and by the method using the linker at the N-terminus. Only small amounts of peptide accumulated at tumor sites. We conclude that phage display is a powerful tool to select peptides with restricted binding specificity; however, the peptides isolated to date do not bind with high retention to tumor sites in vivo.     

Human breast tumor imaging using 111In labeled monoclonal antibody: Athymic mouse model

The monoclonal antibody (MoAb) 323/A3, an IgG1, was raised against the human breast tumor cell line MCF-7 and recognized a 43 Kd membrane associated glycoprotein. Histochemical studies with the antibody detected 75% of metastatic lymph nodes, 59% of primary breast tumors, and showed some staining in 20% of benign breast lesions. For radionuclide imaging, the MoAb 323/A3 was labeled with both ¹²⁵I and ¹¹¹In, via covalently coupled diethylenetriaminepentaacetic acid (DTPA) by the mixed anhydride method. The antibody activity of the DTPA modified 323/A3 was assessed by an immunoassay using viable and fixed MCF-7 target cells. Male athymic nude mice bearing BT-20 human mammary tumors were injected with dual ¹²⁵I/¹¹¹In labeled DTPA 323/A3 via the tail veins. The animals were imaged with a gamma camera equipped with a pinhole collimator at 1–3 h, 1, 2, 3, 4 and 5 days after the tracer administration. On day 5 or 6, the animals were killed, and the biodistribution of the radiotracers was determined for the blood, thyroid, heart, lungs, liver, spleen, kidneys, gastrointestinal tract and tumor. Target to blood ratio at 6 days for the ¹¹¹In tracer was 24:1 in the group with a mean tumor weight of 0.492 g, and 13:1 in another group with a mean tumor weight of 0.1906 g (day 5). However, the ¹²⁵I activity showed only 3.6:1 and 5.4:1 target to blood ratios in the corresponding groups. The larger tumors localized less ¹¹¹In tracer (27.13%±7.57% injected dose/g, Mean±SD) than the smaller tumors (52.75%±22.25% ID/g). Analysis of the gamma images showed that the maximum tracer concentration occurred in the tumors at about 2 to 3 days after intravenous tracer administration. The excellent tumor resolution observed with BT-20 tumors may be due to increased 43 Kd glycoprotein antigen density in this tumor cell line.     

Characterization,biodistribution and small-animal SPECT of I-125-labeled c-Met binding peptide in mice bearing c-Met receptor tyrosine kinase-positive tumor xenografts

c-Met is a receptor tyrosine kinase involved in tumor cell growth, invasion, metastases and angiogenesis. Overexpression of c-Met is frequently observed in several tumor types. Here, we report the in vitro cell-binding properties and biodistribution and SPECT/CT imaging in glioma (U87MG) xenograft-bearing mice of ¹²⁵I-labeled c-Met-binding peptides (cMBPs) including analogs conjugated to amino acid and aliphatic carbon linkers. In vitro assays showed that the peptide without any linker and those with GGG and 8-aminooctanoic acid linkers had low cellular internalization and that IC₅₀ values of peptides were 1.5 μM, 65 nM and 85.3 nM, respectively. Biodistribution studies showed the GGG-containing peptide had higher tumor uptake and a higher tumor-to-blood activity concentration ratio than other receptor-binding ligands. SPECT/CT studies with a dedicated small-animal imaging system were performed in U87MG-bearing athymic mice. Although U87MG tumor xenografts could be visualized by SPECT/micro-CT using the various ¹²⁵I labeled cMBPs, image contrast and overall quality were unremarkable.     

Comparisons of labeling efficiency,biological activity and biodistribution among 125I-, 67Ga-DTPA-and 67Ga-DFO-lectins

The labeling efficiency, biological activity and biodistribution of ¹²⁵I labeled and ⁶⁷Ga chelating agent conjugated lectins were investigated. Pisum sativum agglutinin (PSA) and Lens culinaris agglutinin (LCH) were efficiently labeled with ⁶⁷Ga using bifunctional chelating agents such as diethylenetriaminepentaacetic acid (DTPA) and deferoxamine (DFO), whereas labeling with ¹²⁵I was significantly less efficient. The agglutinating activity of these lectins towards Ehrlich ascites tumor (EAT) cells was retained on conjugation with DFO, but not with DTPA. The in vitro binding ratio of ⁶⁷Ga-DFO-lectins for EAT cells was almost the same as that of ¹²⁵I-lectins. However, the value was significantly decreased in the case of ⁶⁷Ga-DTPA-lectins. In the biodistribution study of radiolabeled lectins in Ehrlich solid tumor (EST) bearing mice, the accumulation of radioactivity in tumor tissue was very much less with ⁶⁷Ga-DTPA-lectins than with ¹²⁵I-lectins. However, the concentration was significantly elevated in the case of ⁶⁷Ga-DFO-lectins. While, these lectins accumulated in liver, spleen, lung, and kidney to a greater extent than ⁶⁷Ga citrate, the tumor to organ ratios became very low. These low tumor to organ ratios, in contrast to ⁶⁷Ga citrate, will certainly inhibit the tumor delineation, and therefore it seems that in spite of a high accumulation ratio of ⁶⁷Ga-DFO-lectins in tumor tissue, these agents are not useful in tumor detection.     

Bifunctional phage-based pretargeted imaging of human prostate carcinoma

IntroductionTwo-step and three-step pretargeting systems utilizing biotinylated prostate tumor-homing bacteriophage (phage) and ¹¹¹In-radiolabeled streptavidin or biotin were developed for use in cancer radioimaging. The in vivo selected prostate carcinoma-specific phage (G1) displaying up to five copies of the peptide IAGLATPGWSHWLAL was the focus of the present study.MethodsThe ability of G1 phage to extravasate and target prostate tumor cells was investigated using immunohistochemistry. G1 phages were biotinylated, streptavidin was conjugated to diethylenetriaminepentaacetic acid (DTPA) and biotin was conjugated to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Biodistribution studies and single-photon emission computed tomography (SPECT)/CT imaging of xenografted PC-3 tumors via two-step pretargeted ¹¹¹In-labeled streptavidin and three-step pretargeted ¹¹¹In-labeled biotin were performed in SCID mice to determine the optimal pretargeting method.ResultsThe ability of G1 phage to extravasate the vasculature and bind directly to human PC-3 prostate carcinoma tumor cells in vivo was demonstrated via immunocytochemical analysis. Comparative biodistribution studies of the two-step and three-step pretargeting strategies indicated increased PC-3 human prostate carcinoma tumor uptake in SCID mice of 4.34±0.26 %ID g^?1 at 0.5 h postinjection of ¹¹¹In-radiolabeled biotin (utilized in a three-step protocol) compared to 0.67±0.06 %ID g^?1 at 24 h postinjection of ¹¹¹In radiolabeled streptavidin (employed in a two-step protocol). In vivo SPECT/CT imaging of xenografted PC-3 tumors in SCID mice with the three-step pretargeting method was superior to that of the two-step pretargeting method, and, importantly, blocking studies demonstrated specificity of tumor uptake of ¹¹¹In-labeled biotin in the three-step pretargeting scheme.ConclusionThis study demonstrates the use of multivalent bifunctional phage in a three-step pretargeting system for prostate cancer radioimaging.     

Residualizing iodine markedly improved tumor targeting using bispecific antibody-based pretargeting.

Previous studies have shown that pretargeting allows rapid visualization of renal cell carcinomas (RCC) with an (111)In-labeled bivalent peptide. For radioimmunotherapy, a beta-emitting radionuclide labeled to a bivalent peptide is required. Therapeutic efficacy of these radionuclides depends on the E(max), physical half-life, and residence time of the radiolabel in the tumor. The (131)I radiolabel generally clears rapidly from the tumor after internalization and subsequent degradation of the bivalent l-amino acid peptide (l-a.a. peptide) in the tumor cells. To improve the residence time of the iodine label in the tumor, a new bivalent peptide was synthesized that is peptidase resistant and consists of 4 d-amino acids (d-a.a. peptide). Here we investigated the characteristics of the residualizing iodine label in SK-RC-52 RCC tumors. METHODS: The d-a.a. peptide was manually synthesized according to standard solid-phase Fmoc/HBTU (2-[1H-benzotriazole-1-yl]-1,1,3,3-tetramethyluronium hexafluorophosphate) chemistry. The uptake and retention in the tumor of (111)In-/(125)I-labeled bivalent peptides (l-a.a. peptide and d-a.a. peptide) were studied in female BALB/c athymic mice with subcutaneous SK-RC-52 RCC tumors. Tumors were pretargeted with the bispecific monoclonal antibody (bs-mAb) G250xDTIn-1 and, 72 h later, mice were injected intravenously with one of both radiolabeled peptides. The effect of bs-mAb-diDTPA-bs-mAb (DTPA is diethylenetriaminepentaacetic acid) bridging at the tumor cell surface on the internalization of the bs-mAb-diDTPA complex was investigated in SK-RC-52 tumor-bearing mice. RESULTS: The maximum uptake and retention of (125)I-labeled l-a.a. peptide in the tumor were significantly lower compared with that of the (111)In-labeled l-a.a. peptide. In contrast, the tumor uptake and retention of the (125)I-labeled d-a.a. peptide) were similar to that of the (111)In-labeled l-a.a. peptide but were superior at later time points. The biodistribution of the radioiodinated d-a.a. peptide was highly similar to that of the (111)In-labeled d-a.a. peptide, and both radiolabeled peptides were retained significantly better in the tumor than the (111)In-labeled l-a.a. peptide. bs-mAb-diDTPA-bs-mAb bridge formation did not affect internalization of the bs-mAb-diDTPA complex. CONCLUSION: Uptake and retention in the tumor of the iodinated peptide after pretargeting with a bs-mAb can be significantly improved using d-a.a. peptides. Accordingly, the radiation dose to the tumor, correlating with the therapeutic efficacy of pretargeted RCC, can be enhanced substantially.     

Tumor uptake of 67Ga-carrying liposomes

The in vivo distribution, excretion, and tumor localization of liposome-encapsulated ⁶⁷Ga in normal and Ehrlich tumor (solid form)-bearing mice were studied. In normal mice, multilamellar vesicles (MLVs) were taken up mainly by the liver and spleen, whereas small unilamellar vesicles (SUVs) exhibited a broader tissue distribution. When ⁶⁷Ga was encapsulated in MLVs or SUVs, the excretion of the radiotracer in the urine and feces was less than that observed for free tracer at 72 h after i.v. administration. In tumor-bearing mice, SUVs were found to accumulate preferentially in tumors. The tumor uptake of neutral, positive, and negative SUVs was 10%–13% of the administered dose per gram of tumor tissue at 24 h after their injection. These values were about three times higher than those found for free ⁶⁷Ga-nitrilotriacetic acid (⁶⁷Ga-NTA) or ⁶⁷Ga-citrate. Significant differences in tumor uptake due to different surface charges of liposomes were not observed. Enhanced tumor-to-blood and tumor-to-muscle ratios were also observed at 24 h after injection. These results suggest that ⁶⁷Ga-carrying liposomes may be a useful for tumor imaging.     

Evaluation of hypoxia-specific cytotoxic bioreductive agent-sodium borocaptate-10B conjugates as 10B-carriers in boron neutron capture therapy

Purpose To evaluate the usefulness of 5 new¹⁰B-compounds (TX-2091, TX-2095, TX-2097, TX-2100, and TX-2110) as¹⁰B-carriers in boron neutron capture therpy (BNCT). They were conjugates that had been synthesized from a hypoxia-specific cytotoxic bioreductive agent, quinoxaline oxide TX-402 and a clinically used¹⁰B-carrier, sodium borocaptate-¹⁰B (BSH). Materials and Methods The 5 new compounds were hybrid compounds that have both a hypoxic cytotoxin unit and a thermal neutron-sensitizing unit, BSH. These new compounds and BSH were administered intraperitoneally to SCC VII tumor-bearing mice. Then, the¹⁰B concentrations in the tumors and normal tissues were measured by γ-ray spectrometry. Subsequently, SCC VII tumor-bearing mice were continuously given 5-bromo-2′-deoxyuridine (BrdU) to label all proliferating (P) cells in the tumors, then treated with TX-2100, which was chosen based on the results of the above-mentioned biodistribution analyses, or BSH in the same manner as in the biodistribution studies. Right after irradiation, during which intratumor¹⁰B concentrations were kept at levels similar to each other, the tumors were excised, minced, and trypsinized. The tumor cell suspensions thus obtained were incubated with cytochalasin-B (a cytokinesis blocker), and the micronucleus (MN) frequency in cells without BrdU labeling [=quiescent (Q) cells] was determined using immunofluorescence staining for BrdU. Meanwhile, the MN frequency in the total (P+Q) tumor cell population was determined from the tumors that were not pretreated with BrdU. Clonogenic cell survival was also determined in mice given no BrdU. Results ¹⁰B biodistribution analyses in tumors, brain, skin, muscles, blood, and liver indicated that TX-2100 has the most favorable characteristics for concentrating a sufficient amount of¹⁰B in tumors and maintaining a high enough¹⁰B concentration during irradiation. In addition, TX-2100 had a significantly stronger radio-sensitizing effect with reactor thermal neutron beams than BSH on both total and Q cells in solid tumors. Further, TX-2100 clearly exhibited a radio-sensitizing effect with γ-rays not only on total cells but also on Q and hypoxic tumor cells, which was not achieved by BSH. Conclusion A¹⁰B-carrier that acts as a hypoxic cytotoxin on tumor cells as well as having the potential to keep¹⁰B in tumors and sensitize tumor cells more markedly than conventional¹⁰B-carriers, such as TX-2100, is a promising candidate for use in BNCT. A part of this paper was presented at the 64th JRS annual meeting in Yokohama on April 8, 2005.     

Effect of chelator conjugation level and injection dose on tumor and organ uptake of 111In-labeled MORAb-009, an anti-mesothelin antibody

Introduction

Radiolabeling of a monoclonal antibody (mAb) with a metallic radionuclide requires the conjugation of a bifunctional chelator to the mAb. The conjugation, however, can alter the physical and immunological properties of the mAb, consequently affecting its tumor-targeting pharmacokinetics. In this study, we investigated the effect of the amount of 2-(p-isothiocyanatobenzyl)-cyclohexyl-diethylenetriamine-pentaacetic acid (CHX-A″) conjugated to MORAb-009, a mAb directed against mesothelin, and the effect of MORAb dose on the biodistribution of ¹¹¹In-labeled MORAb-009.

Methods

We used nude mice bearing the A431/K5 tumor as a mesothelin-positive tumor model and the A431 tumor as a mesothelin-negative control. To find the optimal level of CHX-A″ conjugation, CHX-A″-MORAb-009 conjugates with 2.4, 3.5 and 5.5 CHX-A″ molecules were investigated. To investigate the effect of injected MORAb-009 dose on neutralizing the shed mesothelin in the circulation, biodistribution studies were performed after the intravenous co-injection of ¹¹¹In-labeled MORAb-009 (2.4 CHX-A″/MORAb-009) with three different doses: 0.2, 2 and 30 μg of MORAb-009.

Results

The tumor uptake in A431/K5 tumor was four times higher than that in A431 tumor, indicating that the tumor uptake in A431/K5 was mesothelin mediated. The conjugate with 5.5 CHX-A″ showed a lower isoelectric point (pI) and lower immunoreactivity (IR) than the 2.4 CHX-A″ conjugate. These differences were reflected in the biodistribution of the ¹¹¹In label. The ¹¹¹In-labeled MORAb-009 conjugated with 2.4 CHX-A″ produced higher tumor uptake and lower liver and spleen uptakes than the 5.5 CHX-A″ conjugate. The biodistribution studies also revealed that the tumor uptake was significantly affected by the injected MORAb-009 dose and tumor size. The 30-μg dose produced higher tumor uptake than the 0.2- and 2-μg doses, whereas the 30-μg dose produced lower liver and spleen uptakes than the 0.2-μg dose.

Conclusion

This study demonstrates that the number of chelate conjugation and the injected dose are two important parameters to achieve high tumor and low non-target organ uptake of ¹¹¹In-labeled MORAb-009. This study also suggests that the injected dose of mAb could be individualized based on the tumor size or the blood level of shed antigen in a patient to achieve the ideal tumor-to-organ radioactivity ratios.     

Evaluation of F-18-labeled 5-iodocytidine (18F-FIAC) as a new potential positron emission tomography probe for herpes simplex virus type 1 thymidine kinase imaging

Objective

Herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene in combination with radiolabeled nucleoside substrates is the most widely used reporter system. This study characterized 1-(2′-deoxy-2′-[¹⁸F]fluoro-β-d-arabinofuranosyl)-5-iodocytosine (¹⁸F-FIAC) as a new potential positron emission tomography (PET) probe for HSV1-tk gene imaging and compared it with 2’-deoxy-2’-[¹⁸F]fluoro-5-iodo-1-β-D-arabinofuranosyluracil (¹⁸F-FIAU) and 2’-deoxy-2’-[¹⁸F]fluoro-5-ethyl-1-β-D-arabinofuranosyluracil(¹⁸F-FEAU) (thymidine analogues) in an NG4TL4-WT/STK sarcoma-bearing mouse model.

Methods

A cellular uptake assay, biodistribution study, radioactive metabolites assay and microPET imaging of NG4TL4-WT/STK tumor-bearing mice post administration of ¹⁸F-FIAC, ¹⁸F-FIAU and ¹⁸F-FEAU were conducted to characterize the biological properties of these tracers.

Results

Highly specific uptake of ¹⁸F-FIAC, ¹⁸F-FIAU and ¹⁸F-FEAU in tk-transfected [tk(+)] cells was observed. The tk(+)-to-tk(−) cellular uptake ratio after a 2-h incubation was 66.6±25.1, 76.3±18.2 and 247.2±37.2, respectively. In biodistribution studies, ¹⁸F-FIAC showed significant tk(+) tumor specificity (12.6; expressed as the tk(+)-to-tk(−) tumor uptake ratio at 2 h postinjection) comparable with ¹⁸F-FIAU (15.8) but lower than ¹⁸F-FEAU (48.0). The results of microPET imaging also revealed the highly specific accumulation of these three radioprobes in the NG4TL4-tk(+) tumor.

Conclusion

Our findings suggested that the cytidine analogue ¹⁸F-FIAC is a new potential PET probe for the imaging of HSV1-tk gene expression. ¹⁸F-FIAC may be regarded as the prodrug of ¹⁸F-FIAU in vivo.     

Evaluation of 64Cu-labeled DOTA-d-Phe1-Tyr3-octreotide (64Cu-DOTA-TOC) for imaging somatostatin receptor-expressing tumors

Objective  In-111 (¹¹¹In)-labeled octreotide has been clinically used for imaging somatostatin receptor-positive tumors, and radiolabeled octreotide analogs for positron emission tomography (PET) have been developed. Cu-64 (⁶⁴Cu; half-life, 12.7 h) is an attractive radionuclide for PET imaging and is produced with high specific activity using a small biomedical cyclotron. The aim of this study is to produce and fundamentally examine a ⁶⁴Cu-labeled octreotide analog, ⁶⁴Cu-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-d-Phe¹-Tyr³-octreotide (⁶⁴Cu-DOTA-TOC). Methods   ⁶⁴Cu produced using a biomedical cyclotron was reacted with DOTA-TOC for 30 min at 45°C. The stability of ⁶⁴Cu-DOTA-TOC was evaluated in vitro (incubated with serum) and in vivo (blood collected after administration) by HPLC analysis. Biodistribution studies were performed in normal mice by administration of mixed solution of ⁶⁴Cu-DOTA-TOC and ¹¹¹In-DOTA-TOC and somatostatin receptor-positive U87MG tumor-bearing mice by administration of ⁶⁴Cu-DOTA-TOC or ⁶⁴Cu-1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid-octreotide (⁶⁴Cu-TETA-OC). The tumor was imaged using ⁶⁴Cu-DOTA-TOC, ⁶⁴Cu-TETA-OC, and FDG with an animal PET scanner. Results   ⁶⁴Cu-DOTA-TOC can be produced in amounts sufficient for clinical study with high radiochemical yield. ⁶⁴Cu-DOTA-TOC was stable in vitro, but time-dependent transchelation to protein was observed after injection into mice. In biodistribution studies, the radioactivity of ⁶⁴Cu was higher than that of ¹¹¹In in all organs except kidney. In tumor-bearing mice, ⁶⁴Cu-DOTA-TOC showed a high accumulation in the tumor, and the tumor-to-blood ratio reached as high as 8.81 ± 1.17 at 6 h after administration. ⁶⁴Cu-DOTA-TOC showed significantly higher accumulation in the tumor than ⁶⁴Cu-TETA-OC. ⁶⁴Cu-DOTA-TOC PET showed a very clear image of the tumor, which was comparable to that of ¹⁸F-FDG PET and very similar to that of ⁶⁴Cu-TETA-OC. Conclusions   ⁶⁴Cu-DOTA-TOC clearly imaged a somatostatin receptor-positive tumor and seemed to be a potential PET tracer in the clinical phase.     

Pharmacokinetics, micro-SPECT/CT imaging and therapeutic efficacy of Re-DXR-liposome in C26 colon carcinoma ascites mice model

The pharmacokinetics and internal radionuclide therapy of intraperitoneally administrated ¹⁸⁸Re-N,N-bis(2-mercaptoethyl)-N′,N′-diethylethylenediamine (BMEDA)-labeled pegylated liposomal doxorubicin (¹⁸⁸Re-DXR-liposome) were investigated in the C26 murine colon carcinoma ascites mouse model. After intraperitoneal administration of the nanotargeted bimodality ¹⁸⁸Re-DXR-liposome, the ascites and tumor accumulation of the radioactivity were observed, the levels of radioactivity within the ascites were maintained at relatively higher levels before 48 h and the levels of radioactivity in the tumor were maintained at steady levels after 4 h. The AUC_(o→∞) of ¹⁸⁸Re-DXR-liposome in blood, ascites and tumor was 9.3-, 4.2- and 4.7-fold larger than that of ¹⁸⁸Re-BMEDA, respectively. The maximum tolerated dose of intraperitoneally administrated ¹⁸⁸Re-DXR-liposome was determined in normal BALB/c mice. The survival, tumor and ascites inhibition of mice after ¹⁸⁸Re-DXR-liposome (22.2 MBq of ¹⁸⁸Re, 5 mg/kg of DXR) treatment were evaluated. Consequently, radiochemotherapeutics of ¹⁸⁸Re-DXR-liposome attained better survival time, tumor and ascites inhibition (decreased by 49% and 91% at 4 days after treatment; P<.05) in mice than radiotherapeutics of ¹⁸⁸Re-liposome or chemotherapeutics of Lipo-Dox did. Therefore, intraperitoneal administration of novel ¹⁸⁸Re-DXR-liposome could provide a benefit and promising strategy for delivery of passive nanotargeted bimodality radiochemotherapeutics in oncology applications.     

<Superscript>18</Superscript>F-Fluoroglucosylation of peptides,exemplified on <Emphasis Type="Italic">cyclo</Emphasis>(RGDfK)

Purpose  Oxime formation between an aminooxy-functionalized peptide and an ¹⁸F-labelled aldehyde has recently been introduced as a powerful method for the rapid one-step chemoselective synthesis of radiofluorinated peptides. Materials and methods  Here, the potential of using routinely produced and thus readily available [¹⁸F]fluorodeoxyglucose ([¹⁸F]FDG) as the aldehydic prosthetic group was investigated using an aminooxyacetyl-conjugated cyclic RGD peptide (cyclo(RGDfK(Aoa-(Boc)) as a model peptide. Results  The use of [¹⁸F]FDG from routine production ([¹⁸F]FDGTUM) containing an excess of d-glucose did not allow the radiosynthesis of [¹⁸F]FDG-RGD in activities >37 MBq in reasonable yield, rendering the direct use of clinical grade [¹⁸F]FDG for the routine clinical synthesis of ¹⁸F-labelled peptides impossible. Using no-carrier-added (n.c.a.) [¹⁸F]FDG obtained via HPLC separation of [¹⁸F]FDGTUM from excess glucose, however, afforded [¹⁸F]FDG-RGD in yields of 56–93% (decay corrected) and activities up to 37 MBq. Suitable reaction conditions were 20 min at 120°C and pH 2.5, and a peptide concentration of 5 mM. In a preliminary in vivo biodistribution study in M21 melanoma-bearing nude mice, [¹⁸F]FDG-RGD showed increased tumour accumulation compared to the “gold standard” [¹⁸F]galacto-RGD (2.18 vs 1.49 %iD/g, respectively, at 120 min after injection), but also slightly increased uptake in non-target organs, leading to comparable tumour/organ ratios for both compounds. Conclusion  These data demonstrate that chemoselective ¹⁸F-labelling of aminooxy-functionalized peptides using n.c.a. [¹⁸F]FDG represents a radiofluorination/glycosylation strategy that allows preparation of ¹⁸F-labelled peptides in high yield with suitable pharmacokinetics. As soon as the necessary n.c.a. preparation of [¹⁸F]FDG prior to reaction with the Aoa-peptide can be implemented in a fully automated [¹⁸F]FDG-synthesis, [¹⁸F]fluoroglucosylation of peptides may represent a promising alternative to currently used chemoselective one-step ¹⁸F-labelling protocols.     

Preparation and biological evaluation of 3-[(76)Br]bromo-α-methyl-L-tyrosine, a novel tyrosine analog for positron emission tomography imaging of tumors

Introduction

3-[¹⁸F]fluoro-α-methyl-l-tyrosine ([¹⁸F]FAMT) is a useful amino acid tracer for positron emission tomography (PET) imaging of malignant tumors. FAMT analogs labeled with ⁷⁶Br, a positron emitter with a long half-life (t_1/2=16.1 h), could potentially be widely used as amino acid tracers for tumor imaging. In this study, 3-[⁷⁶Br]bromo-α-methyl-l-tyrosine ([⁷⁶Br]BAMT) was designed, and its usefulness was evaluated as a novel PET tracer for imaging malignant tumors.

Methods

In this study, both [⁷⁶Br]BAMT and [⁷⁷Br]BAMT were prepared. The in vitro and in vivo stability of [⁷⁷Br]BAMT was evaluated by HPLC analysis. Cellular uptake and retention of [⁷⁷Br]BAMT and [¹⁸F]FAMT were evaluated using LS180 colon adenocarcinoma cells. Biodistribution studies were performed in normal mice and in LS180 tumor-bearing mice, and the tumors were imaged with a small-animal PET scanner.

Results

[⁷⁷Br]BAMT was stable in vitro but was catabolized after administration in mice. Cellular accumulation and retention of [⁷⁷Br]BAMT were significantly higher than those of [¹⁸F]FAMT. In biodistribution studies, the tumor accumulation of [⁷⁷Br]BAMT was higher than that of [¹⁸F]FAMT. However, some level of debromination was seen, which caused more retention of radioactivity in the blood and organs than was seen with [¹⁸F]FAMT. PET imaging with [⁷⁶Br]BAMT enabled clear visualization of the tumor, and the whole-body image using [⁷⁶Br]BAMT was similar to that using [¹⁸F]FAMT.

Conclusions

[⁷⁷Br]BAMT showed high levels of tumor accumulation, and [⁷⁶Br]BAMT enabled clear visualization of the tumor by PET imaging. Although an improvement in stability is still needed, ⁷⁶Br-labeled FAMT analogs could potentially serve as PET tracers for the imaging of malignant tumors.     

Rapid synthesis and in vitro and in vivo evaluation of folic acid derivatives labeled with fluorine-18 for PET imaging of folate receptor-positive tumors

In an attempt to visualize folate receptors that overexpress on many cancers, [¹⁸F]-fluorobenzene and pyridinecarbohydrazide-folate/methotrexate conjugates ([¹⁸F]-1, [¹⁸F]-2-folates and [¹⁸F]-8, [¹⁸F]-9-MTXs) were synthesized by the nucleophilic displacement reactions using ethyl-trimethylammonium-benzoate and pyridinecarboxylate precursors. The intermediates ethyl [¹⁸F]-fluorinated benzene and pyridine esters were reacted with hydrazine to produce the [¹⁸F]-fluorobenzene and pyridinecarbohydrazides, followed by coupling with N-hydroxysuccinimide-folate/MTX. Radiochemical yields were greater than 80% (decay corrected), with total synthesis time of less than 45 min. Radiochemical purities were always greater than 97% without high-performance liquid chromatography purification. These synthetic approaches hold considerable promise as rapid and simple method for the radiofluorination of folate derivatives with high radiochemical yield in short synthesis time. In vitro tests on KB cell line showed that significant amount of the radioconjugates were associated with cell fractions, and in vivo characterization in normal Balb/c mice revealed rapid blood clearance of these radioconjugates with excretion predominantly by the urinary and partially by the hepatobiliary systems. Biodistribution studies in nude mice bearing human KB cell line xenografts demonstrated significant tumor uptake and favorable biodistribution profile for [¹⁸F]-2-folate over the other conjugates. The uptake in the tumors was blocked by excess coinjection of folic acid, suggesting a receptor-mediated process. Micro-positron emission tomography images of nude mice bearing human KB cell line xenografts confirmed these observations. These results demonstrate that [¹⁸F]-2-folate may be useful as molecular probe for detecting and staging of folate receptor-positive cancers, such as ovarian cancer and their metastasis as well as monitoring tumor response to treatment.     

153Sm-DTPA-c(CGRRAGGSC)对人MHCC97-H肝癌裸鼠模型的抑瘤作用

目的 探讨¹⁵³Sm-DTPA-c(CGRRAGGSC)对人MHCC97-H肝癌裸鼠模型移植瘤生长的抑制作用.方法 采用间接法合成¹⁵³Sm-DTPA-c(CGRRAGGSC)放射性药物.对8种不同细胞系IL-11受体表达,进行Western blot分析,选择肿瘤接种.20只MHCC97-H皮下荷瘤裸鼠按随机数字表法分为4组(对照组,低、中、高剂量组),每组5只.低、中、高剂量组尾静脉分别注入¹⁵³Sm-DTPA-c(CGRRAGGSC),剂量依序为5.5、11.0和22.0 MBq/0.2 ml,对照组尾静脉注入生理盐水0.2 ml.观察16 d后处死,比较肿瘤的体积,计算抑瘤率;观测标本病理改变;免疫组织化学检测5.5 MBq低剂量组瘤体组织治疗后Ki-67、Bcl-2和IL-11受体表达改变;Western blot检测不同剂量组IL-11受体的变化情况.结果 选择IL-11受体表达最高的MHCC97-H细胞接种.尾静脉内注射¹⁵³Sm-DTPA-c(CGRRAGGSC)后,早期瘤体中央可出现坏死、干瘪、结痂,后期又出现坏死周围瘤组织继续增长.低、中、高剂量组抑瘤率分别为(22.72±2.76)%、(34.65±2.36)%和(85.13±5.78)%(F＝89.32, P<0.05).免疫组织化学显示,对照组及低剂量组瘤内IL-11受体阳性率分别为(84.13±5.71)%和(61.57±5.98)%(t=13.62, P<0.05),低剂量组瘤细胞排列相对疏松,细胞内染色数量明显减少.低剂量组Ki-67、Bcl-2阳性率均较对照组下降(t=20.91、6.68, P<0.05).Western blot结果显示,随抑瘤作用增加,IL-11受体蛋白表达降低.结论 ¹⁵³Sm-DTPA-c(CGRRAGGSC)能够有效抑制人MHCC97-H肝癌裸鼠模型移植瘤的生长,促进IL-11受体表达下调及诱导凋亡作用.     

Application of lectins to tumor imaging radiopharmaceuticals

We investigated the in vitro binding of ¹²⁵I-lectins to Ehrlich ascites tumor (EAT) cells and in vivo uptake of ¹²⁵I-lectins in Ehrlich solid tumor (EST) bearing mice. In in vitro binding assays, phaseolus vulgaris agglutinin (PHA), pisum sativum agglutinin (PSA), and concanavalia agglutinin (Con A) showed a high affinity for EAT cells. The in vivo biodistribution of ¹²⁵I-lectins showed ¹²⁵I-PSA to be significantly taken up into EST tissues 24 h postinjection. After IV injection of ¹²⁵I-PSA, uptake of the radioactivity into the tumor tissues reached a maximum at 6 h, and thereafter decreased. Rapid clearance of the radioactivity from blood and its exretion into kidney soon after injection of ¹²⁵I-PSA were observed. When compared with the biodistribution of ⁶⁷Ga-citrate in EST bearing mice 24 h postinjection, tumor to liver (T/B), tumor to muscle (T/M), and tumor to blood (T/B) ratios were superior for ¹²⁵I-PSA. At 6 h postinjection, the T/B-ratio of ¹²⁵I-PSA was 2.5, and this value may be sufficient to enable discernable diagnostic images. Our results suggest that PSA might be a useful tumor imaging radiopharmaceutical.     

Anticancer activity of targeted proapoptotic peptides.

Tumor-induced angiogenesis can be targeted by RGD (Arg-Gly-Asp) peptides, which bind to alpha(v)beta(3)-receptors upregulated on angiogenic endothelial cells. RGD-containing peptides are capable of inducing apoptosis through direct activation of procaspase-3 to caspase-3 in cells. Additionally, tumor cells overexpressing somatostatin receptors can be targeted by somatostatin analogs. Radiolabeled somatostatin analogs are successfully used to image and treat such tumors via receptor-targeted scintigraphy and therapy. We combined these 2 peptides, RGD and somatostatin, to synthesize a new hybrid peptide, RGD-diethylenetriaminepentaacetic acid (DTPA)-octreotate (c(Arg-Gly-Asp-D-Tyr-Asp)-Lys(DTPA)-D-Phe-c(Cys-Tyr-D-Trp-Lys-Thr-Cys)-Thr). An earlier study showed that tumor-bearing rats had high receptor-specific uptake of RGD-(111)In-DTPA-octreotate in somatostatin receptor subtype 2-positive tissues and tumors. Furthermore, RGD-(111)In-DTPA-octreotate showed a pronounced tumoricidal effect, which is probably the result of increased apoptosis, as is shown by an increased caspase-3 activity after incubation with (111)In-labeled RGD-DTPA-octreotate in comparison with the 2 monopeptides (111)In-DTPA-RGD and (111)In-DTPA-Tyr(3)-octreotate. In this study, we evaluated the biodistributions of RGD-(111)In-DTPA-octreotate and (125)I-RGD-octreotate and investigated the caspase-3 activation of the unlabeled compound RGD-DTPA-octreotate in vitro. METHODS: Biodistribution studies on tumor-bearing rats were performed with RGD-(111)In-DTPA-octreotate and (125)I-RGD-octreotate. The apoptotic activity, by activation of caspase-3 with RGD-DTPA-octreotate and RGD-octreotate, was examined using colorimetric and immunocytochemical assays. RESULTS: The radiolabeled compound, RGD-(111)In-DTPA-octreotate, showed high uptake and retention in the rats in which rat pancreatic CA20948 tumor had been implanted. A major drawback was high renal uptake. In vitro, the unlabeled peptide RGD-DTPA-octreotate induced a significant increase in caspase-3 levels in various cell lines in comparison with RGD and Tyr(3)-octreotate (P < 0.01). Caspase-3 activation was time dependent. To alter the elimination route, we examined the biodistribution of radioiodinated RGD-octreotate without DTPA [c(Arg-Gly-Asp-D-Tyr-Asp)-D-Phe-c(Cys-Tyr-D-Trp-Lys-Thr-Cys)-Thr], as a model of unlabeled RGD-octreotate, in tumor-bearing rats. (125)I-RGD-octreotate showed a much lower renal uptake than did RGD-(111)In-DTPA-octreotate. Furthermore, the affinity of RGD-octreotate increased in comparison with RGD-DTPA-octreotate (values of 1.4 x 10(-8) mol/L vs. 9.4 x 10(-8) mol/L, respectively, for inhibitory concentration of 50%). Finally, RGD-octreotate was still able to activate caspase-3, as was indicated with immunocytochemistry. CONCLUSION: Because of the high renal uptake, RGD-(111)In-DTPA-octreotate is unsuitable for radionuclide therapy. However, the unlabeled peptides, RGD-DTPA-octreotate and RGD-octreotate, also induced an increase in caspase-3 levels, indicating the therapeutic potential of this compound. Thus, the development of hybrid molecules can become a new approach in the treatment of cancer.     

Technetium thiodiglycollic acid (99mTc-TDG). A new radiopharmaceutical with the potential for the assessment of renal function

A mixture of hydrophilic complexes is formed on the reduction (employing tin metal as a reductant) of sodium pertechnetate ^99mTc in the presence of the ligand thiodiglycollic acid (TDG). When administered to rats, the mixture demonstrated a renal clearance rate marginally greater than Glomerular filtration rate (GFR). HPLC analysis indicated the formation of two technetium complexes of TDG. After isolation of the complexes and their administration to rats, one (complex 1) showed renal clearance similar to that of ^99mTc Diethylenetriamine pentaacetic acid (DTPA), while the other (complex 2) demonstrated renal clearance similar to that of ¹²⁵I o-iodo hippuric acid. On heating the mixture of complexes, the proportion of the faster-clearing ^90mTc TDG complex increased to 92% of the total activity, and the biodistribution of the material following heat rearrangement was equivalent to that of isolated complex 2.     

Tumor targeting and SPECT imaging properties of an 111In-labeled galectin-3 binding peptide in prostate carcinoma

IntroductionGalectin-3 (gal-3) is a carbohydrate binding protein that has been implicated in cell adhesion, tumor invasion and metastasis. The objective of this study was to evaluate the tumor targeting and imaging properties of a gal-3 binding peptide selected by phage display in a mouse model of metastatic human prostate carcinoma expressing gal-3.MethodsA gal-3 binding peptide, ANTPCGPYTHDCPVKR, was synthesized with a Gly–Ser–Gly (GSG) spacer and 1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA) and then radiolabeled with ¹¹¹In. The in vitro cell binding properties of ¹¹¹In-DOTA-(GSG)-ANTPCGPYTHDCPVKR were determined in metastatic human PC3-M prostate carcinoma cells. The pharmacokinetics and single-photon emission computed tomographic (SPECT/CT) imaging with the radiolabeled peptide were evaluated in SCID mice bearing human PC3-M prostate carcinoma tumor xenografts.ResultsThe radiolabeled peptide bound with a 50% inhibitory concentration of 191±10.2 nM to cultured PC3-M prostate carcinoma cells. In vivo tumor uptake and retention coupled with fast whole-body clearance of the peptide were demonstrated in PC3-M tumor-bearing SCID mice. The tumor uptake rates of the radiolabeled peptide were 1.27±0.10%ID/g at 30 min, 0.82±0.15%ID/g at 1 h and 0.57±0.09%ID/g at 2 h. MicroSPECT/CT studies revealed good tumor uptake of ¹¹¹In-DOTA-(GSG)-ANTPCGPYTHDCPVKR 2 h postinjection, while uptake in normal organs was low, with the exception of the kidneys.ConclusionsIn vitro cell binding along with tumor uptake of ¹¹¹In-DOTA-(GSG)-ANTPCGPYTHDCPVKR in PC3-M human prostate carcinoma tumor-bearing SCID mice suggests the potential of this peptide as a radiopharmaceutical for imaging of gal-3-expressing prostate tumors.