Sodium iodide symporter (NIS)-mediated radiovirotherapy of hepatocellular cancer using a conditionally replicating adenovirus.

In this study, we determined the in vitro and in vivo efficacy of sodium iodide symporter (NIS) gene transfer and the therapeutic potential of oncolytic virotherapy combined with radioiodine therapy using a conditionally replicating oncolytic adenovirus. For this purpose, we used a replication-selective adenovirus in which the E1a gene is driven by the mouse alpha-fetoprotein (AFP) promoter and the human NIS gene is inserted in the E3 region (Ad5-E1/AFP-E3/NIS). Human hepatocellular carcinoma cells (HuH7) infected with Ad5-E1/AFP-E3/NIS concentrated radioiodine at a level that was sufficiently high for a therapeutic effect in vitro. In vivo experiments demonstrated that 3 days after intratumoral (i.t.) injection of Ad5-E1/AFP-E3/NIS HuH7 xenograft tumors accumulated approximately 25% ID g(-1) (percentage of the injected dose per gram tumor tissue) (123)I as shown by (123)I gamma camera imaging. A single i.t. injection of Ad5-E1/AFP-E3/NIS (virotherapy) resulted in a significant reduction of tumor growth and prolonged survival, as compared with injection of saline. Combination of oncolytic virotherapy with radioiodine treatment (radiovirotherapy) led to an additional reduction of tumor growth that resulted in markedly improved survival as compared with virotherapy alone. In conclusion, local in vivo NIS gene transfer using a replication-selective oncolytic adenovirus is able to induce a significant therapeutic effect, which can be enhanced by additional (131)I application.

Alpha-fetoprotein promoter-targeted sodium iodide symporter gene therapy of hepatocellular carcinoma.

Due to limited treatment options the prognosis of patients with advanced hepatocellular cancer (HCC) has remained poor. To investigate an alternative therapeutic approach, we examined the feasibility of radioiodine therapy of HCC following human sodium iodide symporter (NIS) gene transfer using a mouse alpha-fetoprotein (AFP) promoter construct to target NIS expression to HCC cells. For this purpose, the murine Hepa 1-6 and the human HepG2 hepatoma cell lines were stably transfected with NIS cDNA under the control of the tumor-specific AFP promoter. The stably transfected Hepa 1-6 cell line showed a 10-fold increase in iodide accumulation, while HepG2 cells accumulated (125)I approximately 60-fold. Tumor-specific NIS expression was confirmed on mRNA level by northern blot analysis, and on protein level by immunostaining, that revealed primarily membrane-associated NIS-specific immunoreactivity. In an in vitro clonogenic assay up to 78% of NIS-transfected Hepa 1-6 and 93% of HepG2 cells were killed by (131)I exposure, while up to 96% of control cells survived. In vivo NIS-transfected HepG2 xenografts accumulated 15% of the total (123)I administered per gram tumor with a biological half-life of 8.38 h, resulting in a tumor absorbed dose of 171 mGy MBq(-1) (131)I. After administration of a therapeutic (131)I dose (55.5 MBq) tumor growth of NIS expressing HepG2 xenografts was significantly inhibited. In conclusion, tumor-specific iodide accumulation was induced in HCC cells by AFP promoter-directed NIS expression in vitro and in vivo, which was sufficiently high to allow a therapeutic effect of (131)I. This study demonstrates the potential of tumor-specific NIS gene therapy as an innovative treatment strategy for HCC.

[Demonstration of angiogenesis and the effect on an antiangiogenetic therapy with 99mTc labeled erythrocytes]. / Darstellung der Angiogenese und der Effekte einer antiangiogenetischen Therapie mit 99mTc-markierten Erythrozyten.

AIM: To evaluate, whether scintigraphic studies with radiolabeled erythrocytes may be used to demonstrate the formation of new vessels during angiogenesis and if an effect on antiangiogenetic therapy could be detected. METHODS: As an angiogenesis model we used the ingrowth of blood vessels in matrigel, subcutaneously injected into mice. In order to measure the relative blood volume in the matrigel non-invasively, mouse erythrocytes were labeled with Technetium-99m DTPA. The amount of activity in the matrigel was measured 30 minutes after injection of the radiolabeled erythrocytes with a gammacamera (in-vivo) and a gammacounter (ex-vivo). These results were correlated with the concentration of hemoglobin in the matrigel and the immunhistochemically evaluated density of blood vessels. The influence of the angiogenesis stimulating growth factor (bFGF) and the antiangiogenetic effect of the cyclooxigenase type 2 inhibitor (COX-2) NS398 were tested. RESULTS: There was a close correlation between the activity concentration in the matrigel and the hemoglobin content. Treatment with bFGF significantly increased the activity concentration from 1.74% +/- ID/g to 4.06% +/- 0.36 (p < 0.01), whereas treatment with NS398 significantly inhibited tracer uptake from 2.83% ID/g +/- 0.33 to 0.87% ID/g +/- 0.12 (p < 0.01). CONCLUSION: These results demonstrate the feasibility of using (99m)Tc labelled erythrocytes for scintigraphic imaging to assess the effects of angiogenesis stimulating and inhibiting interventions non-invasively.

[Intravesical radioimmunotherapy of carcinoma in situ of the urinary bladder after BCG failure]. / Intravesikale Radioimmuntherapie des Carcinoma in situ der Harnblase nach BCG-Versagen.

BACKGROUND: In failure to respond to bacillus Calmette-Guérin (BCG) in patients with carcinoma in situ (CIS) of the urinary bladder, radical cystectomy remains the mainstay after BCG failure. OBJECTIVES: The aim of this pilot study was to evaluate tolerability and safety of the α­emitter radioimmunoconjugate instillation in patients after BCG failure. MATERIALS AND METHODS: Nine patients were included. After emptying the bladder via a transurethral catheter, Bi-213-anti-EGFR-mAb was instilled. Treatment was terminated by emptying of the radioimmunoconjugate from the bladder 120 min after instillation. Efficacy was evaluated via endoscopy and histology 6 weeks after instillation. RESULTS: All patients showed excellent toleration of the treatment without any side effects. Treatment resulted in complete eradication of tumor cells in 3 patients and persistent tumor detection in the other 6 patients. CONCLUSIONS: Intravesical instillation of Bi-213-anti-EGFR-mAb is a promising therapeutic option for treatment of in situ bladder cancer after BCG failure for patients who wish to preserve the bladder.

The sensitivity of the alkaline comet assay in detecting DNA lesions induced by X rays, gamma rays and alpha particles.

Experiments were designed and performed in order to investigate whether or not the different cellular energy deposition patterns of photon radiation with different energies (29 kV, 220 kV X rays; Co-60, Cs-137-gamma-rays) and alpha-radiation from an Am-241 source differ in DNA damage induction capacity in human cells. For this purpose, the alkaline comet assay (single cell gel electrophoresis) was applied to measure the amount of DNA damage in relation to the dose received. The comet assay data for the parameters '% DNA in the tail' and 'tail moment' for human peripheral lymphocytes did not indicate any difference in the initial radiation damage produced by 29 kV X rays relative to the reference radiations, 220 kV X rays and the gamma rays, whether for the total mean dose range of 0-3 Gy nor in the low-dose range. In contrast, when the 'tail length' data were analysed saturation of the fitted dose response curve appeared for X rays at about 1.5 Gy but was not apparent for gamma rays up to 3 Gy. Preliminary data for alpha exposures of HSC45-M2 cells showed a significant increase in DNA damage only at high doses (>2 Gy Am-241), but the damage at 2 Gy exceeded the damage induced at 2 Gy by Cs-137-gamma-rays by a factor of 2.5. In contrast, other experiments involving different cell systems and DNA damage indicators such as chromosomal aberrations have detected a significant increase in DNA damage at much lower doses, that is at 0.02 Gy for Am-241 and depicte a higher biological effectiveness. These results indicate that differences in biological effects arise through downstream processing of complex DNA damage.

Therapeutic effect of m-[131I]- and m-[125I]iodobenzylguanidine on neuroblastoma multicellular tumor spheroids of different sizes.

m-[l25I]iodobenzylguanidine (m-[125I]MIBG) has been suggested as an alternative to m-[131I]MIBG for the treatment of metastatic neuroblastoma to achieve a higher radiation dose in micrometastases. To compare these two radiopharmaceuticals, a mathematical model was developed in the present study that allows for the calculation of radiation dose rates within small spherical tumors for different distributions of 131I and 125I. Furthermore, the relationship between tumor size and the therapeutic effects of m-[131I]- and m-[125I]MIBG was studied in vitro using multicellular tumor spheroids of the neuroblastoma cell line SK-N-SH. According to the calculations, higher mean dose rates can be achieved by m-[125I]MIBG than by m-[131I]MIBG up to a tumor diameter of 100 microm when both substances are homogeneously distributed within the tumor. In larger tumors, however, mean dose rates achieved by 131I are up to 8-fold higher. Evaluation of various activity distributions demonstrated that even in tumors of less than 100 microm in diameter, marked heterogeneities of the dose rate can occur when m-[125I]MIBG is not distributed homogeneously. By treatment with m-[131I]MIBG, the growth of tumor spheroids ranging from 100 to 250 microm in diameter was inhibited more effectively in the larger than in the smaller spheroids. The growth inhibition of spheroids treated with m-[125I]MIBG was independent of the spheroid size. In consistency with the calculations, the therapeutic effect of m-[125I]- and m-[131I]MIBG was equal in spheroids with diameters of about 100 microm. In larger spheroids, m-[131I]MIBG induced a more pronounced delay in spheroid growth than m-[125I]MIBG. According to these calculations and in vitro data, m-[125I]MIBG as a single agent does not seem to be a promising alternative to m-[131I]MIBG for treatment of metastatic neuroblastoma. However, the combined use of m-[131I]- and m-[125I]MIBG may be more effective than treatment with m-[131I]MIBG alone.

Noninvasive imaging of alpha(v)beta3 integrin expression using 18F-labeled RGD-containing glycopeptide and positron emission tomography.

The alpha(v)beta3 integrin is an important cell adhesion receptor involved in tumor-induced angiogenesis and tumor metastasis. Here we describe the 18F-labeling of the RGD-containing glycopeptide cyclo(-Arg-Gly-Asp-D-Phe-Lys(sugar amino acid)-) with 4-nitrophenyl 2-[18F]fluoropropionate and the evaluation of this compound in vitro and in tumor mouse models. Binding assays with isolated immobilized alpha(v)beta3, alpha(v)beta5, and alpha(IIb)beta3 as well as in vivo studies using alpha(v)beta3-positive and -negative murine and xenotransplanted human tumors demonstrated receptor-specific binding of the radiolabeled glycopeptide yielding high tumor:background ratios (e.g., 120 min postinjection: tumor:blood, 27.5; tumor:muscle, 10.2). First imaging results using a small animal positron emission tomograph suggest that this compound is suitable for noninvasive determination of the alpha(v)beta3 integrin status and therapy monitoring.

Highly specific tumor binding of a 213Bi-labeled monoclonal antibody against mutant E-cadherin suggests its usefulness for locoregional alpha-radioimmunotherapy of diffuse-type gastric cancer.

A monoclonal antibody (E-cadherin delta 9-1) directed against a characteristic E-cadherin mutation (in-frame deletion of exon 9), found in diffuse-type gastric cancer but not in any normal tissue, was conjugated with the high linear energy transfer alpha-emitter 213Bi and tested for its binding specificity in s.c. and i.p. nude mice tumor models. After intratumoral application in s.c. tumors expressing mutant E-cadherin, the 213Bi-labeled antibody was specifically retained at the injection site as shown by autoradiography. After injection into the peritoneal cavity, uptake in small i.p. tumor nodules expressing mutant E-cadherin was 17-fold higher than in tumor nodules expressing wild-type E-cadherin (62% injected dose/g versus 3.7% injected dose/g). 78% of the total activity in the ascites fluid was bound to free tumor cells expressing mutant E-cadherin, whereas in control cells, binding was only 18%. The selective binding of the 213Bi-labeled, mutation-specific monoclonal antibody E-cadherin delta 9-1 suggests that it will be successful for alpha-radioimmunotherapy of disseminated tumors after locoregional application.

Non-invasive imaging of implanted peritoneal carcinomatosis in mice using PET and bioluminescence imaging.

BACKGROUND: Non-invasive imaging of peritoneal carcinomatosis remains challenging. The aim of this study was to compare positron emission tomography (PET) and bioluminescence imaging (BLI) for the early detection of peritoneal carcinomatosis in a mouse model. METHODS: Female nude mice were inoculated intraperitoneally with 1×10(7) HSC45-M2-luc gastric cancer cells. The cells were stably transfected with the gene coding for firefly luciferase. Tumour development was monitored using PET and BLI and in two subgroups, on days 3 and 4 or on days 6 and 7 after tumour cell inoculation. Tumour nodules found on post mortem examination served as the reference standard for evaluating the images. RESULTS: PET detected 58/82 lesions (sensitivity 71 %). This method detected all (100 %) nodules larger than 6 mm, 88 % of nodules in the range of >2-4 mm, and even 58 % of small nodules measuring only 1-2 mm. BLI identified a total of 40/82 lesions (sensitivity 49 %). The difference between PET and BLI was statistically significant at p < 0.05 (PET/BLI chi-square 8.2). CONCLUSIONS: PET was more sensitive than BLI for the detection of early peritoneal carcinomatosis in our mouse model. The sensitivity of BLI largely depended on the site of the lesions in relation to the imaging device.

Radiolabeled thymidine: a sensitive tracer for early tumor response and recurrence after irradiation.

UNLABELLED: This study evaluated the sensitivity of a radiolabeled thymidine tracer for assessment of early tumor response and recurrence after irradiation. METHODS: SW707 human colon carcinoma implanted into nude mice was irradiated with 6 or 20 Gy. Tumor volume was determined for an interval of 14 d. At 4, 8 and 24 h and at 2, 3, 7, 10 and 14 d after irradiation, [14C]thymidine uptake into the tumor was determined with a liquid scintillation counter and the intratumoral distribution of [14C]thymidine was visualized and evaluated semiquantitatively by autoradiography using a phosphor imager. RESULTS: In both groups, tumor volume decreased until day 7 after irradiation; afterward, regrowth occurred in only the group that had received 6 Gy. A decrease in thymidine uptake was found as early as 8 h after irradiation. On day 3 after irradiation, thymidine uptake increased again in the 6-Gy group, before the increase in tumor volume, but remained unchanged in the 20-Gy group. Also on day 3, multiple foci of thymidine uptake suggesting proliferation preceding tumor recurrence were seen on autoradiographs from the 6-Gy group but not from the 20-Gy group. Histological findings correlated with the results of autoradiography. CONCLUSION: The results show that radiolabeled thymidine is a sensitive tracer for assessment of early tumor response and recurrence after irradiation. The rapid decrease in uptake, however, does not allow any prediction about tumor recurrence.

Tumor cell spheroids as a model for evaluation of metabolic changes after irradiation.

UNLABELLED: Tumor cell spheroids provide a good model to evaluate the relationship between tumor volume and the number of viable cells in the volume with the uptake of metabolic tracers before and after therapy. They represent the only in vitro model that allows the determination of the activity per unit volume, a parameter which is relevant for interpretation of PET studies. The purpose of this study was to evaluate this model with respect to the uptake of 14C-FDG, 3H-methionine and 3H-thymidine with and without exposure to irradiation. METHODS: Spheroids of the human adenocarcinoma cell line SW 707 were incubated in media containing 14C-FDG, 3H-methionine or 3H-thymidine for 1 hr at 1, 4, 8, 24 and 48 hr after exposure to a single radiation dose of 6 Gy together with control spheroids. Tracer uptake after incubation was expressed in cpm/ spheroid, cpm/1000 viable cells and cpm/0.01 mm3. In addition, the proliferative capacity of control and irradiated spheroids was determined using the clonogenic assay. RESULTS: Spheroid uptake of FDG decreased with time after irradiation, while the uptake per 1000 viable cells was increased significantly. The activity per unit volume remained unchanged in comparison to control spheroids. Methionine uptake per spheroid was unchanged after irradiation because of the high increase in uptake per 1000 viable cells. Uptake per unit volume also remained unchanged in comparison to controls. Thymidine uptake per 1000 viable cells did not change after irradiation but showed significant differences in uptake per spheroid and per unit volume compared to controls. The percentage of thymidine incorporated into the TCA-precipitable fraction containing DNA was 50% in controls and decreased to 12% at 24 hr after irradiation. The suppressed clonogenic capacity early after therapy recovered with the increase in thymidine uptake and with the increase in thymidine incorporation into DNA. CONCLUSION: The results show that the activity determined within a certain tumor volume is a balance between the increased tracer uptake by surviving cells after therapy and the lack of tracer uptake by dead cells, which still contribute to the tumor volume. Thus, the resulting unchanged activity per unit volume within the spheroid, as found for FDG and methionine, may not fully reflect therapy-induced metabolic changes in tumors.

Investigation of transport mechanism and uptake kinetics of O-(2-[18F]fluoroethyl)-L-tyrosine in vitro and in vivo.

UNLABELLED: The aim of the study was to investigate the transport mechanism and uptake kinetics of the new 18F-labeled amino acid O-(2-[18F]fluoroethyl)-L-tyrosine (L-[18F]FET) and D-[18F]FET in human SW 707 colon carcinoma cells and the in vivo biodistribution of this tracer in SW 707 tumor-bearing mice. METHODS: SW 707 cells were incubated with L- and D-[18F]FET under physiologic amino acid concentrations with and without the competitive transport inhibitors 2-amino-2 norbornane-carboxylic acid and a-(methylamino)isobutyric acid plus serine. For the investigation of the transport capacity, unlabeled L-FET was added to the samples. In addition, xenotransplanted mice were injected intravenously with L-[18F]FET; killed 10, 30, 60 and 120 min after injection; and the radioactivity concentration in different organs was measured in a gamma counter. RESULTS: The in vitro kinetic experiments showed a fast initial uptake of L-[18F]FET into the cells up to 6 min, followed by a nearly constant tracer concentration. The accumulation factor, calculated as the ratio between intracellular and extracellular tracer concentration, ranged from 3.0 to 5.0. In comparison, D-[18F]FET did not accumulate in the cells. Washing the cells in medium at 37 degrees C, after a 30-min incubation with L-[F-18]FET, led to a rapid decrease of radioactivity, which demonstrates the bidirectional transport. In addition, experiments with increasing concentrations of unlabeled L-FET indicated a linear correlation between L-FET uptake rate and the extracellular concentration. Results of transport inhibition experiments with the specific competitive inhibitors demonstrated that the uptake of L-FET into SW 707 cells was caused mainly (>80%) by the transport system L. In the in vivo studies, the half-life (t1/2 beta) of L-[18F]FET in the plasma was determined to be 94 min and the uptake into the brain increased to 120 min with a brain-to-blood ratio of 0.86. The xenotransplanted tumor showed higher uptake of L-[18F]FET (>6 %ID/g) at 30 and 60 min than all other organs, except the pancreas. The tumor-to-blood ratio reached about 2 between 30 and 120 min. CONCLUSION: L-[18F]FET, which is transported by the specific amino acid transport system L, seems to be a potential amino acid tracer for tumor imaging and therapy monitoring with PET.

Synthesis and radiopharmacology of O-(2-[18F]fluoroethyl)-L-tyrosine for tumor imaging.

UNLABELLED: The aim of the study was to develop a simple 18F-labeled amino acid as a PET tracer for cerebral and peripheral tumors. O-(2-[18F]fluoroethyl)-L-tyrosine (L-[18F]FET) was synthesized and biologically evaluated. Results of the first human PET study are reported. METHODS: No carrier added (n.c.a.) and D-[18F]FET were prepared by 18F-fluoroethylation of L- and D-tyrosine in a two-step procedure. Biodistribution studies were performed in mice. The metabolic fate of L-[18F]FET was investigated in plasma, brain, tumor and pancreatic tissue samples using chromatographic procedures. Tumor uptake studies were performed in mammary carcinoma-bearing mice and in mice with the colon carcinoma SW 707. In a human PET study, a 59-y-old man with a recurrent astrocytoma was imaged using n.c.a. L-[18F]FET. RESULTS: Synthesis of [18F]FET was accomplished in about 50 min with an overall radiochemical yield of 40%. The uptake of L-[18F]FET in the brain of mice reached a level >2% ID/g between 30 and 60 min postinjection. The brain uptake of the D-isomer was negligible, indicating blood-brain barrier penetration by a specific amino acid transport system. L-[18F]FET is not incorporated into proteins. High-performance liquid chromatography (HPLC) analysis of brain, pancreas and tumor homogenates as well as plasma samples of mice at 10, 40 or 60 min postinjection showed only unchanged L-[18F]FET. Activity uptake in the bone did not exceed 2% ID/g at 40 min postinjection. The brain uptake of L-[18F]FET in mice bearing mammary carcinomas and colon carcinomas reached 7.1%+/-1.2% ID/g and 6.4%+/-1.7% ID/g 1h postinjection, respectively. In the first human study, L-[18F]FET-PET allowed a clear delineation of a recurrent astrocytoma. Thirty-five minutes postinjection, the tumor-to-cortex ratio was >2.7. A tumor-to-blood ratio >1.5 was reached at 30 min postinjection and continued to increase. No significant activity accumulation was observed in peripheral organs after approximately 40 min postinjection. CONCLUSION: The high in vivo stability of L-[18F]FET, its fast brain and tumor uptake kinetics, its low accumulation in nontumor tissue and its ease of synthesis strongly support further evaluation of L-[18F]FET as an amino acid tracer for cerebral and peripheral tumors.

Comparison of sestamibi, tetrofosmin, and Q12 retention in porcine myocardium.

UNLABELLED: Although there are several 99mTc perfusion tracers introduced for clinical use, there are no data available directly comparing these tracers with microsphere-determined flow. The aim of this study was to compare the myocardial retention of sestamibi, tetrofosmin, and Q12 in a porcine model. METHODS: We used a pig model with (n = 6) or without (n = 3) coronary occlusion. Each pig received a simultaneous injection of sestamibi and either tetrofosmin (group 1, n = 5) or Q12 (group 2, n = 4) labeled with either 99mTc or 95mTc (physical half-life, 61 d; photon energy, 204 keV) during pharmacologic vasodilation. Absolute myocardial retention of each tracer was calculated from the myocardial tracer activity and arterial input function. RESULTS: The plot of all three tracers versus flow achieved a plateau at a higher flow range. However, sestamibi showed a higher mean retention than either tetrofosmin (group 1, 0.27 +/- 0.11 vs. 0.16 +/- 0.06 mL/g/min, respectively; P < 0.01) or Q12 (group 2, 0.32 +/- 0.13 vs. 0.09 +/- 0.03 mL/g/min, respectively; P < 0.01). Furthermore, when a linear regression analysis was performed to assess the relationship between retention and microsphere-determined flow, sestamibi showed a greater increment in retention than did tetrofosmin or Q12. CONCLUSION: Although all of the tracers showed a nonlinear increase in retention as flow increased, sestamibi may display more favorable characteristics as a flow tracer in the porcine heart.

Glycosylated RGD-containing peptides: tracer for tumor targeting and angiogenesis imaging with improved biokinetics.

UNLABELLED: The alpha(v)beta3 integrin plays an important role in metastasis and tumor-induced angiogenesis. Targeting with radiolabeled ligands of the alpha(v)beta3 integrin may provide information about the receptor status and enable specific therapeutic planning. Previous studies from our group resulted in tracers that showed alpha(v)beta3-selective tumor uptake. However, these first-generation compounds predominantly revealed hepatobiliary excretion with high radioactivity found in the liver. In this report, the synthesis and biological evaluation of the first glycosylated RGD-containing peptide (RGD-peptide) for the noninvasive imaging of alpha(v)beta3 expression are described. METHODS: Peptides were assembled on a solid support using fluorenylmethoxycarbonyl-coupling protocols. The precursor cyclo(-Arg-Gly-Asp-D-Tyr-Lys(SAA)-) GP1 was synthesized by coupling 3-acetamido-2,6-anhydro-4,5,7-tri-O-benzyl-3-deoxy-beta-D-glycero-D-gulo-heptonic acid (SAA(Bn3)) with cyclo(-Arg(Mtr)-Gly-Asp(OtBu)-D-Tyr(tBu)-Lys-) and subsequent removal of the protection groups. Iodine labeling was performed by the Iodo-Gen method (radiochemical yield > 50%). The in vitro binding assays were performed using purified immobilized alpha(IIb)beta3, alpha(v)beta5, and alpha(v)beta3 integrins. For in vivo experiments, nude mice bearing xenotransplanted melanomas and mice with osteosarcomas were used. RESULTS: The glycosylated peptide 3-iodo-Tyr4-cyclo(-Arg-Gly-Asp-D-Tyr-Lys(SAA)-) GP2 showed high affinity and selectivity for alpha(v)beta3 in vitro (50% inhibitory concentration = 40 nmol/L). Pretreatment studies indicate specific binding of [125I]GP2 on alpha(v)beta3-expressing tumors in vivo. Comparison of the pharmacokinetics of [125I]GP2 and [125I]-3-iodo-Tyr4-cyclo(-Arg-Gly-Asp-D-Tyr-Val-) [125I]P2 revealed for [125I]GP2 an increased activity concentration in the blood (e.g., 3.59 +/- 0.35 percentage injected dose [%ID]/g vs. 1.72 +/- 0.44 %ID/g at 10 min postinjection) and a significantly reduced uptake in the liver (e.g., 2.59 +/- 0.24 %ID/g vs. 21.96 +/- 2.78 %ID/g at 10 min postinjection). Furthermore, a clearly increased activity accumulation in the tumor was found (e.g., 3.05 +/- 0.31 %ID/g vs. 0.92 +/- 0.16 %ID/g at 240 min postinjection), which remained almost constant between 60 and 240 min postinjection. This resulted in good tumor-to-organ ratios for the glycosylated tracer (e.g., 240-min postinjection osteosarcoma model: tumor-to-blood = 16; tumor-to-muscle = 7; tumor-to-liver = 2.5), which were confirmed by the first gamma-camera images of osteosarcoma-bearing mice at 240 min postinjection. CONCLUSION: This study demonstrates that the introduction of a sugar moiety improves the pharmakokinetic behavior of a hydrophobic peptide-based tracer. Additionally, this alpha(v)beta3-selective glycosylated radioiodinated second-generation tracer GP2 shows high tumor uptake and good tumor-to-organ ratios that allow noninvasive visualization of alpha(v)beta3-expressing tumors and monitoring therapy with alpha(v)beta3 antagonists. Finally, the favorable biokinetics make the glycosylated RGD-peptide a promising lead structure for tracers to quantify the alpha(v)beta3 expression using PET.

Radiolabeled alpha(v)beta3 integrin antagonists: a new class of tracers for tumor targeting.

UNLABELLED: The alpha(v)beta3 integrins play an important role during tumor metastasis and tumor-induced angiogenesis. Targeting of this receptor may provide information about the receptor status of the tumor and enable specific therapeutic planning. Cyclo(-Arg-Gly-Asp-D-Phe-Val-) has been shown to be a selective alpha(v)beta3 integrin antagonist with high affinity. In this study we describe the synthesis and biological evaluation of [125I]-3-iodo-D-Tyr4-cyclo(-Arg-Gly-Asp-D-Tyr-Val-) ([125I]P2), [125I]-3-iodo-Tyr5-cyclo(-Arg-Gly-Asp-D-Phe-Tyr-) ([125I]P4) and the negative control peptide [1251]-3-iodo-D-Tyr4-cyclo(-Arg-D-Ala-Asp-Tyr-Val-) ([125I]P6). METHODS: Peptides were assembled on a solid support using fluorenylmethoxycarbonyl amino acid coupling protocols. Radioiodination was performed using the iodogen method. The in vitro binding assays were performed using isolated, immobilized alphaIIbeta3 and alpha(v)beta3 integrins. Expression of the alphaVbeta3 receptor on the different tumors was validated by immunohistochemical methods using alpha(v) and alpha(v)beta3 specific antibodies. For biodistribution studies, nude mice with melanoma M21 or mammary carcinoma MaCaF and BALB/c mice with osteosarcoma were used. RESULTS: The in vitro binding assays demonstrate that the introduction of tyrosine and subsequent iodination have no influence on the high affinity and selectivity for alpha(v)beta3. Immunohistochemical staining clearly indicates the presence of the alpha(v)beta3 integrins on the tumor tissue of the melanoma and the osteosarcoma. Pretreatment and displacement studies show specific binding of [125I]P2 on melanoma M21-bearing nude mice and osteosarcoma-bearing BALB/c mice but less specific binding on mammary carcinomas. [125I]P2 exhibits fast elimination kinetics. The accumulation in the tumor 10 min postinjection is 2.07 +/- 0.32 %ID/g for the melanoma M21 and 3.50 +/- 0.49 %ID/g for the osteosarcoma and decreases to 1.30 +/- 0.13 %ID/g and 2.03 +/- 0.49 %ID/g 60 min postinjection, respectively. [125I]P4 shows even faster elimination kinetics, resulting in a tumor accumulation of 0.40 +/- 0.10 %ID/g 60 min postinjection for the osteosarcoma-bearing BALB/c mice. Both peptides reveal predominately hepatobiliary excretion. For [1251]P2, this also is confirmed by autoradiography. The negative control peptide [125I]P6 shows no specific activity accumulation. CONCLUSION: [125I]P2 exhibits high affinity and selectivity for the alpha(v)beta3 integrin in vitro and in vivo and, thus, represents the first radiolabeled alpha(v)beta3 antagonist for the investigation of angiogenesis and metastasis in vivo.

Preclinical evaluation of 4-[18F]fluoroprolines: diastereomeric effect on metabolism and uptake in mice.

The aim of this study was to evaluate the diastereomeric effect on uptake and metabolic behavior of (2S,4R)-4-[18F]fluoro-L-proline (trans-[18F]FPro) and (2S,4S)-4-[18F]fluoro-L-proline (cis-[18F]FPro) in view of their potential suitability as tracers for abnormal collagen synthesis. No-carrier-added 4-[18F]fluoro-L-prolines were prepared according to the literature in about 150 min (50-60% radiochemical yield). Both compounds exhibited high in vivo stability. The tumor uptake of cis-[18F]FPro in osteosarcomas of mice was high and at 240 min postinjection reached 11.8 +/- 2.2 %ID/g compared with 7.07 +/- 1.68 %ID/g for trans-[18F]FPro. In contrat to trans-[18F]FPro, which showed fast and complete renal clearance, the cis isomer was accumulated in the pancreas, and showed hepatic clearance and renal reuptake. Speciation studies on tissue homogenates revealed protein incorporation only for cis-[18F]FPro. However, due to the relatively slow protein incorporation rate of cis-[18F]FPro, the tumor uptake of both compounds in colon carcinomas, mammary carcinomas, and osteosarcomas 1 h postinjection predominantly reflect amino acid transport.

Targeting of gelatinase activity with a radiolabeled cyclic HWGF peptide.

Matrix metalloproteinases (MMPs) are a family of proteinases that play an important role in cancer as well as in numerous diseases. In this article, we describe the labeling of a phage display selected cyclic decapeptide containing the HWGF (histidine-tryptophane-glycine-phenylalanine) sequence to target MMP-2 and MMP-9. To evaluate the ability of this labeled peptide to monitor non invasively MMP-2 and MMP-9 activity, in vitro studies, biodistribution, competition studies and plasma metabolites analyses in Lewis Lung cancer tumor bearing mice were performed.

Distribution of disialoganglioside GD2-antigen and binding of anti-GD2 antibodies on spheroids of neuroblastoma cell line.

The ganglioside GD2 is highly expressed on human tumors of neuroectodermal origin. We investigated by scanning electron microscopy the ganglioside GD2 distribution on the surface of spheroids of the neuroblastoma cell line SK-N-LO. About 50% of cells showed GD2 on the plasma membrane and the distribution of GD2 on most of these cells was heterogeneous, with more GD2 at the contact sites of the cells. The binding kinetics of the chimeric anti-GD2 antibody ch14.18 labelled with I-125 on spheroids (average diameter: 450 microns) was determined by gamma counting. Over 4 hours the antibody concentration was raised substantially but up to 24 hours there was only a very slow further increase. A clustered pattern of bound chimeric anti-GD2 antibody ch14.18 was found on a cross-section of the spheroid by autoradiography.

Growth inhibition of human papillary thyroid carcinoma cells and multicellular spheroids by anti-EGF-receptor antibody.

EGF has been reported to stimulate thyroid cell proliferation. In the present study we investigated the effects of anti-EGF-R-antibody (Mab 4253 both as monolayers and spheroids of an oxyphilic, non iodine metabolizing, papillary thyroid carcinoma cell line (ONCO-DG-1) and roughly characterized their EGF-R. Scatchard analysis with I-125-labeled-EGF demonstrated a low number of 1.5 x 10(4) EGF-R per monolayer cell (KD 4.1 X 10(-10) M) and only 6 x 10(3) EGF-R per spheroids cell (KD 5.0 X 10(-10) M). Already 80% of the binding sites were blocked by only 0.44 microgram/ml Mab 425. Proliferathe activity and EGF-R were found to be regularly distributed throughout the spheroids. Adding Mab 425 to medium containing 1 ng/ml EGF, inhibited the growth of monolayer cells by 15% (1 ng/ml Mab 425) and 28% (10 ngiml Mab 425), measured by the MTT-test. The volume growth of spheroids was inhibited by 10-15% using 2 micrograms/ml Mab 425, whereas their viability (MTT-Test) was almost identical. The results show that the anti-EGF-R-antibody (Mab 425) alone is not effective enough for therapeutical use, but it could be of clinical value in conjugation with radionuclides (e.g. I-131) in order to reach metastases not metabolizing iodine.