The αvβ3 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 αvβ3, αvβ5, and αIIbβ3 as well as in vivo studies using αvβ3-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 αvβ3 integrin status and therapy monitoring.

Numerous studies have shown that the integrin αvβ3 is an important receptor affecting tumor growth, local invasiveness, and metastatic potential (1). This dimeric transmembrane glycoprotein mediates adhesion and migration of tumor cells on a variety of extracellular matrix proteins. Furthermore, αvβ3 is strongly expressed on activated endothelial cells and plays a critical role in the angiogenic process (2). In contrast, expression of αvβ3 is weak in resting endothelial cells and most normal organ systems. Thus, inhibition of αvβ3 is currently being evaluated as a new strategy for tumor-specific anticancer therapy. Similar to several other integrins, αvβ3 recognizes the tripeptide sequence arginine-glycine-aspartic acid (RGD). The affinity of integrins toward different ligands is critically determined by the conformation of this common binding motif. Thus, the design of RGD-containing peptides with the corresponding conformation allows selective targeting of specific integrins. In previous studies (3, 4), we used spatial screening techniques for the development of the first αv-selective inhibitor, cyclo(-Arg-Gly-Asp-d-Phe-Val-) (5). More recently, peptidomimetic αvβ3 antagonists have been developed (6, 7). Inhibition of αvβ3 function by these peptidic and nonpeptidic antagonists has been shown to inhibit tumor growth in animal studies (6, 7, 8). Future developments of anti-αvβ3-directed therapy and translation of these encouraging experimental data to clinical studies would be greatly facilitated by noninvasive techniques that allow serial studies of αvβ3-positive tumors. In an animal model, Sipkins et al.(9) recently demonstrated that it is feasible to image αvβ3 expression using magnetic resonance imaging and antibody-coated paramagnetic liposomes.

The aim of this study was to develop a radiolabeled analogue of cyclo(-Arg-Gly-Asp-d-Phe-Val-) that is suitable for imaging of αvβ3 expression using PET.3 In addition, the feasibility of small animal PET systems for monitoring blockade of αvβ3 by specific antagonists was evaluated in living mice.

All organic reagents were purchased from Merck KGaA (Darmstadt, Germany), Aldrich (Steinheim, Germany), or Fluka (Neu-Ulm, Germany) and used without further purification. Human M21 and M21-L melanoma cells were kindly provided by Dr. D. A. Cheresh (Departments of Immunology and Vascular Biology, The Scripps Research Institute, La Jolla, CA). Analytical as well as preparative RP-HPLC was performed on Sykam equipment (Gilching, Germany) using a YMC-Pack J’sphere H80 (4 μm; 150 × 20 mm) column (YMC Co., Ltd., Kyoto, Japan). For radioactivity measurements, the outlet of the UV detector was connected to a well-type NaI(Tl) detector from EG&G (München, Germany).

Preparation of [18F]Galacto-RGD.

The synthesis of the Fmoc-protected SAA (7-amino-l-glycero-l-galacto-2,6-anhydro-7-deoxyheptanoic acid) and the fluorination precursor [cyclo(-Arg-Gly-Asp-d-Phe-Lys(SAA)-)] as well as the reference glycopeptide will be described elsewhere.4 N.c.a. [18F]fluoride (t1/2 =109.7 min) was produced via the 18O(p,n)18F nuclear reaction by bombardment of an isotopically enriched [18O]water target with an 11 MeV proton beam using the RDS-112 cyclotron (Siemens/CTI, Knoxville, TN). The glycopeptide was labeled using n.c.a. 4-nitrophenyl 2-[18F]fluoropropionate (specific activity, approximately 70 TBq/mmol), which was prepared as described by Wester et al.(10). Briefly, 6 μmol of cyclo(-Arg-Gly-Asp-d-Phe-Lys(SAA)-) were dissolved in 150 μl of DMSO and added to an Eppendorf cap with dried 4-nitrophenyl 2-[18F]fluoropropionate (approximately 185 MBq). After this, 30 μmol of potassium salt of 1-hydroxy-benzotriazole in 50 μl of DMSO were added and allowed to stand for 15 min at 70°C. Isolation of the 18F-labeled glycopeptide was carried out using RP-HPLC with an acetonitrile/water/0.1% trifluoroacetic acid gradient (10–50% acetonitrile in 20 min; flow rate, 10 ml/min, tR = 11.6 min, K′ = 5.1). The solvent was removed in vacuo, and the residue was dissolved in PBS (pH 7.4) to obtain solutions that were ready for use in animal experiments.

Biological Assay.

Purification of the proteins and the isolated integrin binding assay have been described elsewhere (11). The inhibitory capacities of [19F]Galacto-RGD were quantified by measuring its effect on the interactions between immobilized integrin and biotinylated soluble ligands (vitronectin or fibrinogen). The integrin preparations differ somewhat over time, thus the αv-selective cyclo(-Arg-Gly-Asp-d-Phe-Val-) was used as internal standard to allow interassay comparability.

Tumor Models.

Biodistribution of [18F]Galacto-RGD was evaluated in mice using a murine osteosarcoma and two xenotransplanted human melanoma models (M21 and M21-L). The osteosarcoma and the M21 melanoma cell line both express the αvβ3 integrin (11, 12). The melanoma M21-L cell line, which was selected for weak expression of the αvβ3 integrin, served as a negative control (12).

Murine osteosarcomas induced by injection of strontium-90 were serially transplanted into female BALB/c mice. Tumor pieces of approximately 1 mm3 were injected by a trocar close to the femur into the musculus quadriceps. Human M21 and M21-L melanoma cells were cultured in a humidified atmosphere with 5% CO2. The cell culture medium was RPMI 1640 (Seromed Biochrom, Berlin, Germany) supplemented with 10% FCS and gentamicin. Tumor xenografts were obtained by s.c. injection of 5 × 106 cells (M21) or 1.5 × 107 cells (M21-L) into the left flank of female nude mice. Mice bearing tumors weighing between 300 and 500 mg were used for biodistribution studies.

Biodistribution Studies.

Nude mice bearing tumor xenografts of human melanoma M21 or M21-L and BALB/c mice bearing murine osteosarcomas were i.v. injected with approximately 370 kBq of [18F]Galacto-RGD. The animals were sacrificed and dissected 10, 60, and 120 min after injection of [18F]Galacto-RGD. Blood, plasma, liver, kidney, muscle, heart, brain, lung, spleen, colon, femur, and tumor were removed and weighed. The radioactivity in the tissue was measured using a gamma counter. Results are expressed as the %ID/g. Each value represents the mean and SD of three to four animals.

Competition Studies.

Blocking of the αvβ3 integrin was completed by injecting 6 mg/kg cyclo(-Arg-Gly-Asp-d-Phe-Val-) 10 min before the injection of 370 kBq of the radioactive compound in 100 μl of PBS (pH 7.4). Animals were sacrificed and dissected 60 min after injection of [18F]Galacto-RGD. Further processing was carried out as described above.

PET Studies with a Dedicated Small Animal Scanner.

PET imaging of tumor-bearing mice was performed using a prototype small animal positron tomograph, Munich Avalanche Photodiode PET (13). The animal scanner consists of two sectors, comprising three detector modules each, which rotate around the animal for acquisition of complete projections in one transaxial slice (30 angular steps). Each module consists of eight small (3.7 × 3.7 × 12 mm3) lutetium-oxy-orthosilicate crystals read out by arrays of avalanche photodiodes. List mode data are reconstructed using statistical, iterative methods including the spatially dependent line spread function. Reconstructed image resolution is 2.5 mm (full width at half maximum) in a transaxial field of view of 7.5 cm, and the slice thickness is 2 mm. Ninety min after the injection of approximately 5.5 MBq of [18F]Galacto-RGD, animals were positioned prone inside the tomograph, and a transaxial slice through the tumor region was measured for 35 min.

One melanoma M21-bearing mouse was imaged three times: (a) without pretreatment; (b) with 6 mg/kg cyclo(-Arg-Gly-Asp-d-Phe-Val-); and (c) with 18 mg/kg cyclo(-Arg-Gly-Asp-d-Phe-Val-) injected 10 min before the tracer. For comparison, one mouse with negative control melanoma M21-L was imaged. Tumor volume was approximately 0.5 ml for both tumor models. To assess tumor uptake of [18F]Galacto-RGD, circular regions of interest with a diameter of 5 mm were placed at the location of the maximum tracer uptake in the tumor and in the contralateral thorax wall (reference region). Relative tracer uptake was expressed as the ratio between mean counts in the tumor and in the reference region (tumor:background ratio).

Labeling of cyclo(-Arg-Gly-Asp-d-Phe-Lys(SAA)-) using 4-nitrophenyl 2-[18F]fluoropropionate as a prosthetic group resulted in [18F]Galacto-RGD (Fig. 1) of high radiochemical purity (>98%). The decay-corrected radiochemical yield for the acylation step after RP-HPLC was approximately 50% within a total preparation time of about 40 min.

The in vitro binding assay showed that the inhibitory peptides were able to fully suppress the binding of the ligands (vitronectin or fibrinogen) to the isolated immobilized receptors (αIIbβ3, αvβ5, and αvβ3) and that the binding kinetics followed a classic sigmoid path. The IC50s of [18F]Galacto-RGD were 5 (αvβ3), 1.000 (αvβ5), and 6.000 nmIIbβ3), respectively. Thus, [18F]Galacto-RGD showed a 200-1200-fold higher affinity for αvβ3 than for αvβ5 and αIIbβ3, respectively.

The biodistribution data of [18F]Galacto-RGD in the three tumor models are summarized in Table 1. [18F]Galacto-RGD showed rapid and predominantly renal excretion, resulting in a low activity concentration in blood and muscle as early as 60 min p.i. The initial activity accumulation in the αvβ3-expressing osteosarcoma and melanoma M21 was between 3% and 4% ID/g 10 min p.i., decreasing to about 1.5% ID/g 60 min p.i. and remaining constant until the end of the observation period. Moreover, at 120 min p.i., most organs showed lower activity uptake like the tumor for both models. Only liver, colon, and kidneys revealed a similar activity concentration to the tumor. The low activity accumulation in the bone indicated that the tracer is stable toward defluorination in vivo. Altogether, this led to high tumor:background ratios [e.g., tumor:blood, 13.2 (osteosarcoma) and 27.5 (M21); tumor:muscle: 6.0 (osteosarcoma) and 10.2 (M21)]. In contrast, the negative control tumors showed an initial tracer uptake of approximately 2% ID/g, which decreased to about 0.4% ID/g after 60 min p.i. Thus, the tracer uptake in the negative control tumor is 3.8 times lower than that in the αvβ3-positive tumor between 60 and 120 min (Fig. 2). Pretreatment of the melanoma M21-bearing mice with 6 mg/kg of the αv-selective peptide cyclo(-Arg-Gly-Asp-d-Phe-Val-) reduced the tumor:blood ratio at 60 min p.i. from 8.7 to 1.5 and the tumor:muscle ratio from 7.4 to 4.2.

The transaxial image (Fig. 3) of a melanoma M21-bearing mouse obtained by the animal scanner demonstrates the high tumor:background ratio found in the biodistribution studies and allowed clear visualization of the αvβ3-expressing tumor in the left flank of the mouse. In contrast, the same experiment using a mouse with the negative control melanoma M21-L on the left flank showed almost no increased activity uptake in the tumor compared to the background (tumor:background, 1.2). Moreover, pretreatment experiments with different amounts of cyclo(-Arg-Gly-Asp-d-Phe-Val-) injected 10 min before the tracer demonstrated dose-dependent blocking of the αvβ3 integrin (Fig. 3). The tumor:background ratio decreased from 5.7 (without pretreatment) to 3.2 [pretreatment with 6 mg/kg cyclo(-Arg-Gly-Asp-d-Phe-Val-)] and 2.1 [pretreatment with 18 mg/kg cyclo(-Arg-Gly-Asp-d-Phe-Val-)].

In this study, we described the first 18F-labeled RGD-containing glycopeptide ([18F]Galacto-RGD) that is suitable for noninvasive imaging of αvβ3 integrin expression using PET. Imaging of αvβ3-positive tumors in living mice using a high-resolution PET scanner resulted in a strong contrast between tumor and normal tissues. Moreover, we demonstrated that serial PET studies using [18F]Galacto-RGD allowed noninvasive assessment of the blockade of the receptor by specific antagonists.

Previously, we introduced radioiodinated cyclic RGD peptides for the imaging of αvβ3 integrin status (11). These first-generation tracers showed receptor-specific accumulation in different tumor mouse models. However, these tracers also revealed high activity retention in liver and intestine, which limits the application for tumor imaging. For the present study, we improved the pharmacokinetics by introducing a SAA, which increased the hydrophilicity and markedly reduced the tracer uptake by the liver. Moreover, introduction of the SAA allows 18F-labeling of the amino methyl function of the glycopeptide using radiolabeled acylation reagents. The design of [18F]Galacto-RGD was based on our data from comprehensive structure activity investigations (3, 4, 5, 14). These studies demonstrated that the specific binding of the cyclic pentapeptides is determined by a “kinked” conformation of the RGD site (see Fig. 1). Introduction of a lysine and subsequent conjugation of the SAA are not expected to change the spatial structure of the peptide. Thus, these modifications are unlikely to influence the αvβ3 affinity of the compound. This was confirmed by the in vitro binding studies, which revealed a high αvβ3 affinity and selectivity for [18F]Galacto-RGD.

Moreover, [18F]Galacto-RGD showed rapid and predominantly renal excretion, resulting in a low tracer concentration in most of the organs (especially blood and muscle) and stable accumulation in the αvβ3-expressing tumor during the observation period (up to 120 min p.i.). These features permit high signal:noise ratios for in vivo imaging soon after injection (2 h), making this tracer suitable for PET studies using short-lived isotopes.

Recently, different nonpeptidic antagonists with high affinity and selectivity have been reported (6, 7). These low molecular mass compounds are optimized in size to fit the binding pocket of the receptor. Thus, introduction of labeling groups is likely to result in a loss of affinity. Sivolapenko et al.(15) described a 99mTc-labeled linear decapeptide containing two RGD sites that had been used for imaging in one patient study. However, neither in vitro nor in vivo studies were carried out to demonstrate αvβ3 affinity and specificity of the peptide. Furthermore, the linear peptide showed high persistent background activity in the lung and abdomen. Most recently, preliminary data have been presented on a 111In-labeled diethylenetriaminepenta-acetic acid-RGD analogue (16), a dimeric, 12-amino acid αvβ3 antagonist labeled with 99mTc or 111In (17), and 99mTc-, 186Re-, and 90Y-labeled peptides based on cyclo(-Arg-Gly-Asp-d-Phe-Lys-) (18). These compounds showed tumor-specific binding in vitro and in vivo.

However, the use of a PET tracer is preferable for imaging receptor expression because of the superior sensitivity and spatial resolution of PET and the ability to quantify regional tracer concentrations. As demonstrated by our study, these principal physical advantages of PET permit imaging of αvβ3 expression in living mice. Furthermore, we were able to demonstrate the feasibility of serial PET studies to determine the specific blockade of αvβ3 after injection of increasing amounts of cyclo(-Arg-Gly-Asp-d-Phe-Val-), a potent αv antagonist. This indicates that [18F]Galacto-RGD may also be used for monitoring anticancer therapy directed at the functional inhibition of αvβ3.

Although these data are very encouraging, the following limitations should also be noted. In this initial evaluation of [18F]Galacto-RGD, αvβ3 receptor density was not quantitatively determined for the tumor models. Furthermore, PET studies were analyzed by simple tumor:background ratios. Quantitative analysis of αvβ3 receptor density by PET imaging will require tracer kinetic analysis and correlation with the expression of the αvβ3 integrin using immunohistochemistry and Western blots. Nevertheless, the uptake of [18F]Galacto-RGD in αvβ3-positive tumors was four times higher than that in negative controls, and a specific antagonist was able to block up to 65% of [18F]Galacto-RGD uptake in receptor-positive tumors. Moreover, as reported previously (11), the nonspecific radiolabeled cyclic pentapeptide 3-[125I]iodo-Tyr4-cyclo(-Arg-d-Ala-Asp-Tyr-Val-) clearly showed lower tumor uptake for both tumor models (osteosarcoma, 0.48 ± 0.39% ID/g at 120 min p.i.; melanoma M21, 0.12 ± 0.04% ID/g at 120 min p.i.). Thus, our findings qualitatively demonstrate receptor specificity of [18F]Galacto-RGD in vivo. In addition, the good correlation between tumor:background ratios determined by small animal PET and invasive biodistribution studies clearly indicate the feasibility of quantitative evaluation of αvβ3 expression by PET imaging.

The use of n.c.a. [18F]Galacto-RGD in humans is not expected to lead to toxicity because plasma concentrations will be in the low nanomolar range. Furthermore, the short physical half-life of 18F (t1/2 = 109.7 min) and rapid renal elimination of unbound tracer result in a low radiation dose. Thus, PET studies using [18F]Galacto-RGD may easily be translated from experimental settings to clinical studies.

Initial clinical trials evaluating the use of αvβ3 antagonists as antiangiogenic therapy in patients with various malignant tumors have been initiated (19). In clinical studies, radiolabeled RGD peptides may be used to document αvβ3 expression of the tumors before the administration of αvβ3 antagonists, thus allowing appropriate selection of patients entering clinical trials. Furthermore, as demonstrated, [18F]Galacto-RGD may be used to assess the inhibition of the αvβ3 integrin by antagonists, a process by which optimization of the dose of αvβ3 antagonists may be achieved. Finally, αvβ3 expression has been reported to be an important factor in determining the invasiveness and metastatic potential of malignant tumors in experimental tumor models as well as in patient studies (20, 21). Therefore, noninvasive imaging of αvβ3 expression using [18F]Galacto-RGD and PET may provide a unique means of characterizing the biological aggressiveness of a malignant tumor in an individual patient.

In conclusion, our study demonstrates that [18F]Galacto-RGD is suitable for imaging of αvβ3 expression using PET. Moreover, we have shown that this tracer, in combination with a small animal PET scanner, may be used to monitor the blockade of αvβ3 by specific antagonists in living mice. We anticipate that noninvasive serial studies of αvβ3 expression and functional activity using PET will become an important tool to evaluate the role of αvβ3 during tumor progression and angiogenesis in basic research as well as in clinical studies.

Fig. 1.

Schematic structure of [18F]Galacto-RGD. The glycopeptide shows the characteristic βII’-turn with d-Phe in the i+1 position and the RGD site in a “kinked” conformation responsible for the αvβ3 selectivity.

Fig. 1.

Schematic structure of [18F]Galacto-RGD. The glycopeptide shows the characteristic βII’-turn with d-Phe in the i+1 position and the RGD site in a “kinked” conformation responsible for the αvβ3 selectivity.

Close modal
Fig. 2.

Comparison of the biodistribution data of [18F]Galacto-RGD in melanoma M21 (▪)- and M21-L (▵)-bearing nude mice. Error bars, 1 SD. For some data points, error bars are not visible because the SD is smaller than the size of the symbol.

Fig. 2.

Comparison of the biodistribution data of [18F]Galacto-RGD in melanoma M21 (▪)- and M21-L (▵)-bearing nude mice. Error bars, 1 SD. For some data points, error bars are not visible because the SD is smaller than the size of the symbol.

Close modal
Fig. 3.

Transaxial small animal PET images of nude mice bearing human melanoma xenografts. Images were acquired 90 min after injection of approximately 5.5 MBq of [18F]Galacto-RGD. The top left image shows selective accumulation of the tracer in the αvβ3-positve (M21) tumor on the left flank. In contrast, no focal tracer accumulation is visible in the αvβ3-negative (M21-L) control tumor (bottom left image). The three images on the right were obtained from serial [18F]Galacto-RGD PET studies in one mouse. These illustrate the dose-dependent blockade of tracer uptake by the αv-selective cyclic pentapeptide cyclo(-Arg-Gly-Asp-d-Phe-Val-).

Fig. 3.

Transaxial small animal PET images of nude mice bearing human melanoma xenografts. Images were acquired 90 min after injection of approximately 5.5 MBq of [18F]Galacto-RGD. The top left image shows selective accumulation of the tracer in the αvβ3-positve (M21) tumor on the left flank. In contrast, no focal tracer accumulation is visible in the αvβ3-negative (M21-L) control tumor (bottom left image). The three images on the right were obtained from serial [18F]Galacto-RGD PET studies in one mouse. These illustrate the dose-dependent blockade of tracer uptake by the αv-selective cyclic pentapeptide cyclo(-Arg-Gly-Asp-d-Phe-Val-).

Close modal

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

Supported by Grant 96.017.2 from the Sander-Foundation and in part by the German Reseach Foundation.

3

The abbreviations used are: PET, positron emission tomography; RP-HPLC, reversed-phase high performance liquid chromatography; SAA, sugar amino acid; Fprop, 2-fluoropropionyl; [18F]Galacto-RGD, cyclo(-Arg-Gly-Asp-d-Phe-Lys(([18F]Fprop)SAA)-); p.i., postinjection; %ID/g, percentage of the injected dose per gram of tissue; n.c.a., non-carrier added.

4

R. Haubner, H. J. Wester, C. Mang, S. L. Goodman, H. Kessler, and M. Schwaiger. Synthesis and in vitro evaluation of a RGD-containing glycopeptide for the noninvasive determination of the αvβ3 expression using PET, manuscript in preparation.

Table 1

Biodistribution data for [18F]Galacto-RGD in melanoma-bearing nude mice and osteosarcoma-bearing mice

Values are given as % ID/g ± SD.
Time p.i. (min) Osteosarcoma (αvβ3 positive) Melanoma M21 (αvβ3 positive) Melanoma M21-L (negative control) 
Blood    
 10 2.58 ± 0.41 2.93 ± 0.53 4.70a 
 60 0.27 ± 0.06 0.18 ± 0.06 0.26 ± 0.06 
 120 0.13 ± 0.03 0.05 ± 0.01 0.13 ± 0.07 
Serum    
 10 5.25 ± 0.33 5.65 ± 0.89 8.71 
 60 0.50 ± 0.12 0.34 ± 0.11 0.49 ± 0.11 
 120 0.19 ± 0.04 0.09 ± 0.01 0.22 ± 0.10 
Liver    
 10 5.47 ± 0.11 5.64 ± 2.27 6.40 
 60 2.94 ± 0.27 1.85 ± 0.39 2.07 ± 0.20 
 120 1.97 ± 0.27 1.25 ± 0.05 1.79 ± 0.58 
Kidneys    
 10 8.67 ± 0.52 8.48 ± 0.84 10.82 
 60 3.11 ± 0.60 1.86 ± 0.10 2.24 ± 0.21 
 120 2.21 ± 0.46 1.52 ± 0.10 1.86 ± 0.46 
Muscle    
 10 0.80 ± 0.13 1.13 ± 0.18 1.44 
 60 0.32 ± 0.06 0.21 ± 0.02 0.21 ± 0.03 
 120 0.28 ± 0.02 0.15 ± 0.02 0.18 ± 0.03 
Spleen    
 10 2.54 ± 0.07 2.10 ± 0.45 2.39 
 60 1.54 ± 0.22 0.69 ± 0.10 0.77 ± 0.02 
 120 1.20 ± 0.04 0.64 ± 0.09 0.73 ± 0.14 
Heart    
 10 1.94 ± 0.05 1.85 ± 0.24 2.02 
 60 0.51 ± 0.07 0.28 ± 0.05 0.34 ± 0.02 
 120 0.63 ± 0.41 0.19 ± 0.04 0.29 ± 0.06 
Tumor    
 10 2.88 ± 0.32 3.90 ± 1.36 1.88 
 60 1.33 ± 0.20 1.56 ± 0.15 0.42 ± 0.09 
 120 1.68 ± 0.49 1.49 ± 0.10 0.44 ± 0.24 
Lung    
 10 3.43 ± 0.24 3.64 ± 0.52 4.84 
 60 1.10 ± 0.18 0.76 ± 0.09 0.83 ± 0.08 
 120 0.72 ± 0.15 0.53 ± 0.05 0.71 ± 0.24 
Femur    
 10 1.22 ± 0.04 1.51 ± 0.34 1.46 
 60 0.54 ± 0.11 0.34 ± 0.05 0.44 ± 0.06 
 120 0.51 ± 0.02 0.33 ± 0.02 0.50 ± 0.10 
Colon    
 10 3.77 ± 0.16 1.74 ± 0.55 1.68 
 60 2.27 ± 1.23 0.57 ± 0.04 0.70 ± 0.19 
 120 1.59 ± 0.46 3.53 ± 0.44 0.76 ± 0.27 
Brain    
 10 0.14 ± 0.01 0.19 ± 0.04 0.19 
 60 0.06 ± 0.01 0.05 ± 0.01 0.05 ± 0.01 
 120 0.05 ± 0.01 0.04 ± 0.01 0.05 ± 0.01 
Values are given as % ID/g ± SD.
Time p.i. (min) Osteosarcoma (αvβ3 positive) Melanoma M21 (αvβ3 positive) Melanoma M21-L (negative control) 
Blood    
 10 2.58 ± 0.41 2.93 ± 0.53 4.70a 
 60 0.27 ± 0.06 0.18 ± 0.06 0.26 ± 0.06 
 120 0.13 ± 0.03 0.05 ± 0.01 0.13 ± 0.07 
Serum    
 10 5.25 ± 0.33 5.65 ± 0.89 8.71 
 60 0.50 ± 0.12 0.34 ± 0.11 0.49 ± 0.11 
 120 0.19 ± 0.04 0.09 ± 0.01 0.22 ± 0.10 
Liver    
 10 5.47 ± 0.11 5.64 ± 2.27 6.40 
 60 2.94 ± 0.27 1.85 ± 0.39 2.07 ± 0.20 
 120 1.97 ± 0.27 1.25 ± 0.05 1.79 ± 0.58 
Kidneys    
 10 8.67 ± 0.52 8.48 ± 0.84 10.82 
 60 3.11 ± 0.60 1.86 ± 0.10 2.24 ± 0.21 
 120 2.21 ± 0.46 1.52 ± 0.10 1.86 ± 0.46 
Muscle    
 10 0.80 ± 0.13 1.13 ± 0.18 1.44 
 60 0.32 ± 0.06 0.21 ± 0.02 0.21 ± 0.03 
 120 0.28 ± 0.02 0.15 ± 0.02 0.18 ± 0.03 
Spleen    
 10 2.54 ± 0.07 2.10 ± 0.45 2.39 
 60 1.54 ± 0.22 0.69 ± 0.10 0.77 ± 0.02 
 120 1.20 ± 0.04 0.64 ± 0.09 0.73 ± 0.14 
Heart    
 10 1.94 ± 0.05 1.85 ± 0.24 2.02 
 60 0.51 ± 0.07 0.28 ± 0.05 0.34 ± 0.02 
 120 0.63 ± 0.41 0.19 ± 0.04 0.29 ± 0.06 
Tumor    
 10 2.88 ± 0.32 3.90 ± 1.36 1.88 
 60 1.33 ± 0.20 1.56 ± 0.15 0.42 ± 0.09 
 120 1.68 ± 0.49 1.49 ± 0.10 0.44 ± 0.24 
Lung    
 10 3.43 ± 0.24 3.64 ± 0.52 4.84 
 60 1.10 ± 0.18 0.76 ± 0.09 0.83 ± 0.08 
 120 0.72 ± 0.15 0.53 ± 0.05 0.71 ± 0.24 
Femur    
 10 1.22 ± 0.04 1.51 ± 0.34 1.46 
 60 0.54 ± 0.11 0.34 ± 0.05 0.44 ± 0.06 
 120 0.51 ± 0.02 0.33 ± 0.02 0.50 ± 0.10 
Colon    
 10 3.77 ± 0.16 1.74 ± 0.55 1.68 
 60 2.27 ± 1.23 0.57 ± 0.04 0.70 ± 0.19 
 120 1.59 ± 0.46 3.53 ± 0.44 0.76 ± 0.27 
Brain    
 10 0.14 ± 0.01 0.19 ± 0.04 0.19 
 60 0.06 ± 0.01 0.05 ± 0.01 0.05 ± 0.01 
 120 0.05 ± 0.01 0.04 ± 0.01 0.05 ± 0.01 
a

Data for this time point result from one mouse.

The RDS-cyclotron team, particularly M. Herz, is acknowledged for routine radionuclide production. We thank W. Linke, S. Daum, F. Rau, and K. Fischer for excellent technical assistance and B. J. Pichler, G. Boening, and M. Rafecas for their contribution in preparing the PET images. We thank Dr. D. A. Cheresh for supplying us with M21 and M21-L melanoma cells.

1
Clezardin P. Recent insights into the role of integrins in cancer metastasis.
Cell. Mol. Life Sci.
,
54
:
541
-548,  
1998
.
2
Stromblad S., Cheresh D. A. Integrins, angiogenesis and vascular cell survival.
Chem. Biol.
,
3
:
881
-885,  
1996
.
3
Gurrath M., Müller G., Kessler H., Aumailley M., Timpl R. Conformation/activity studies of rationally designed potent anti-adhesive RGD peptides.
Eur. J. Biochem.
,
210
:
911
-921,  
1992
.
4
Haubner R., Finsinger D., Kessler H. Stereoisomeric peptide libraries and peptidomimetics for designing selective inhibitors of the αvβ3 integrin for a new cancer therapy.
Angew. Chem. Int. Ed. Engl.
,
36
:
1374
-1389,  
1997
.
5
Aumailley M., Gurrath M., Müller G., Calvete J., Timpl R., Kessler H. Arg-Gly-Asp constrained within cyclic pentapeptides. Strong and selective inhibitors of cell adhesion to vitronectin and laminin fragment P1.
FEBS Lett.
,
291
:
50
-54,  
1991
.
6
Carron C. P., Meyer D. M., Pegg J. A., Engleman V. W., Nickols M. A., Settle S. L., Westlin W. F., Ruminski P. G., Nickols G. A. A peptidomimetic antagonist of the integrin αvβ3 inhibits Leydig cell tumor growth and the development of hypercalcemia of malignancy.
Cancer Res.
,
58
:
1930
-1935,  
1998
.
7
Kerr J. S., Wexler R. S., Mousa S. A., Robinson C. S., Wexler E. J., Mohamed S., Voss M. E., Devenny J. J., Czerniak P. M., Gudzelak A., Jr., Slee A. M. Novel small molecule αv integrin antagonists: comparative anti-cancer efficacy with known angiogenesis inhibitors.
Anticancer Res.
,
19
:
959
-968,  
1999
.
8
Lode H. N., Moehler T., Xiang R., Jonczyk A., Gillies S. D., Cheresh D. A., Reisfeld R. A. Synergy between an antiangiogenic integrin αv antagonist and an antibody-cytokine fusion protein eradicates spontaneous tumor metastases.
Proc. Natl. Acad. Sci. USA
,
96
:
1591
-1596,  
1999
.
9
Sipkins D. A., Cheresh D. A., Kazemi M. R., Nevin L. M., Bednarski M. D., Li K. C. P. Detection of tumor angiogenesis in vivo by αvβ3-targeted magnetic resonance imaging.
Nat. Med.
,
5
:
623
-626,  
1998
.
10
Wester H. J., Hamacher K., Stöcklin G. A comparative study of n.c.a. fluorine-18 labeling of proteins via acylation and photochemical conjugation.
Nucl. Med. Biol.
,
23
:
365
-372,  
1996
.
11
Haubner R., Wester H. J., Reuning U., Senekowitsch-Schmidtke R., Diefenbach B., Kessler H., Stöcklin G., Schwaiger M. Radiolabeled αvβ3 integrin antagonists: a new class of tracers for tumor targeting.
J. Nucl. Med.
,
40
:
1061
-1071,  
1999
.
12
Cheresh D. A., Spiro R. C. Biosynthetic and functional properties of an Arg-Gly-Asp-directed receptor involved in human melanoma cell attachment to vitronectin, fibrinogen, and von Willebrand factor.
J. Biol. Chem.
,
262
:
17703
-17711,  
1987
.
13
Ziegler S. I., Pichler B. J., Boening G., Rafecas M., Pimpl W., Lorenz E., Schmitz N., Schwaiger M. A prototype high resolution animal positron tomograph with avalanche photodiode arrays and LSO crystals.
Eur. J. Nucl. Med.
,
28
:
136
-143,  
2001
.
14
Haubner R., Gratias R., Diefenbach B., Goodman S. L., Jonczyk A., Kessler H. Structural and functional aspects of RGD-containing cyclic pentapeptides as highly potent and selective integrin αvβ3 antagonists.
J. Am. Chem. Soc.
,
118
:
7461
-7472,  
1996
.
15
Sivolapenko G. B., Skarlos D., Pectasides D., Stathopoulou E., Milonakis A., Sirmalis G., Stuttle A., Courtenay-Luck N. S., Konstantinides K., Epenetos A. A. Imaging of metastatic melanoma utilising a technetium-99m-labelled RGD-containing synthetic peptide.
Eur. J. Nucl. Med.
,
25
:
1383
-1389,  
1998
.
16
De Jong M., Van Hagen P. M., Breeman W. A., Bernard H. F., Schaar M., Van Gameren A., Srinivasan A., Schmidt M., Bugaj J. E., Krenning E. P. Evaluation of a radiolabeled cyclic DTPA-RGD analog for tumor imaging and radionuclide therapy.
J. Nucl. Med.
,
41
:
232P
2000
.
17
Janssen M. L. H., Boerman O. C., Edwards D. S., Barnett J. A., Rajopadhye W. J., Oyen G., Corstens F. H. M. 111In- and 99mTc-labeled peptides against the αvβ3 integrin: a new target for radionuclide peptide targeting of tumors.
J. Nucl. Med.
,
41
:
33P
2000
.
18
Bock M., Bruchertseifer F., Haubner R., Senekowitsch-Schmidtke R., Kessler H., Schwaiger M., Wester H. J. Tc-99m-, Re-188- and Y-90-labeled αvβ3 antagonists: promising tracer for tumor-induced angiogenesis.
J. Nucl. Med.
,
41
:
41P
2000
.
19
Brower V. Tumor angiogenesis-new drugs on the block.
Nat. Biotechnol.
,
17
:
963
-968,  
1999
.
20
Felding-Habermann B., Mueller B. M., Romerdahl C. A., Cheresh D. A. Involvement of integrin αv gene expression in human melanoma tumorigenicity.
J. Clin. Investig.
,
89
:
2018
-2022,  
1992
.
21
Gasparini G., Brooks P. C., Biganzoli E., Vermeulen P. B., Bonoldi E., Dirix L. Y., Ranieri G., Miceli R., Cheresh D. A. Vascular integrin αvβ3: a new prognostic indicator in breast cancer.
Clin. Cancer Res.
,
4
:
2625
-2634,  
1998
.