In vivo evaluation and small-animal PET/CT of a prostate cancer mouse model using 64Cu bombesin analogs: side-by-side comparison of the CB-TE2A and DOTA chelation systems.

The BB2 receptor subtype, of the bombesin family of receptors, has been shown to be highly overexpressed in a variety of human tumors, including prostate cancer. Bombesin (BBN), a 14-amino acid peptide, has been shown to target the BB2 receptor with high affinity. 64Cu (half-life = 12.7 h, beta+: 18%, E(beta+ max) = 653 keV; beta-: 37%, E(beta- max) = 578 keV) is a radioisotope that has clinical potential for application in both diagnostic imaging and radionuclide therapy. Recently, new chelation systems such as 1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid (CB-TE2A) have been reported to significantly stabilize the 64Cu radiometal in vivo. The increased stability of the 64Cu-CB-TE2A chelate complex has been shown to significantly reduce nontarget retention compared with tetraazamacrocycles such as 1,4,7,10-tetraazacyclodoadecane-N,N',N',N'-tetraacetic acid (DOTA). The aim of this study was to determine whether the CB-TE2A chelation system could significantly improve the in vivo stability of 64Cu bombesin analogs. The study directly compares 64Cu bombesin analogs using the CB-TE2A and DOTA chelation systems in a prostate cancer xenograft SCID (severely compromised immunodeficient) mouse model. METHODS: The CB-TE2A-8-AOC-BBN(7-14)NH2 and DOTA-8-AOC-BBN(7-14)NH2 conjugates were synthesized and radiolabeled with 64Cu. The receptor-binding affinity and internalization profile of each metallated conjugate was evaluated using PC-3 cells. Pharmacokinetic and small-animal PET/CT studies were performed using female SCID mice bearing PC-3 xenografts. RESULTS: In vivo BB2 receptor targeting was confirmed by tumor uptake values of 6.95 +/- 2.27 and 4.95 +/- 0.91 %ID/g (percentage injected dose per gram) at the 15-min time point for the 64Cu-CB-TE2A and 64Cu-DOTA radioconjugates, respectively. At the 24-h time point, liver uptake was substantially reduced for the 64Cu-CB-TE2A radioconjugate (0.21 +/- 0.06 %ID/g) compared with the 64Cu-DOTA radioconjugate (7.80 +/- 1.51 %ID/g). The 64Cu-CB-TE2A-8-AOC-BBN(7-14)NH2 radioconjugate demonstrated significant clearance, 98.60 +/- 0.28 %ID, from the mouse at 24 h after injection. In contrast, only 67.84 +/- 5.43 %ID of the 64Cu activity was excreted using the 64Cu-DOTA-8-AOC-BBN(7-14)NH2 radioconjugate because of nontarget retention. CONCLUSION: The pharmacokinetic and small-animal PET/CT studies demonstrate significantly improved nontarget tissue clearance for the 64Cu-CB-TE2A8-AOC-BBN(7-14)NH2. This is attributed to the improved in vivo stability of the 64Cu-CB-TE2A chelate complex as compared with the 64Cu-DOTA chelate complex.     

Gallium-68-labeled DOTA-rhenium-cyclized alpha-melanocyte-stimulating hormone analog for imaging of malignant melanoma

INTRODUCTION: Diagnosis of malignant melanoma is critical, since a patient's prognosis is poor. Previous studies have shown that 64Cu- and 86Y-DOTA-ReCCMSH(Arg11) have the potential for early detection of malignant melanoma by exploiting the sensitivity and high resolution of positron emission tomography (PET). This encouraged us to investigate DOTA-ReCCMSH(Arg11) labeled with another beta+-emitting radionuclide, 68Ga. METHODS: DOTA-ReCCMSH(Arg11) was successfully labeled with 68Ga at pH 3.8-4 at 85 degrees C. Acute biodistribution and small-animal PET imaging studies were performed in mice bearing B16/F1 melanoma tumor. RESULTS: Biodistribution studies showed moderate receptor-mediated tumor uptake, fast nontarget organ clearance and high tumor to nontarget tissue ratios. Preadministration of d-lysine significantly reduced kidney uptake without affecting the uptake of the agent in the tumor. Small-animal PET images showed that the tumor could be clearly visualized at all time points examined (0.5-2 h) with the standardized uptake value analysis following a similar trend as the biodistribution data. CONCLUSIONS: The preliminary data obtained suggest that 68Ga-DOTA-ReCCMSH(Arg11) is a promising PET imaging agent for early detection of malignant melanoma.     

A gallium-labeled DOTA-alpha-melanocyte- stimulating hormone analog for PET imaging of melanoma metastases.

Although (18)F-FDG PET is widely used for metastatic melanoma diagnosis, it is less accurate than desirable, particularly for small foci. Since both melanotic and amelanotic melanomas overexpress receptors for alpha-melanocyte-stimulating hormone (alpha-MSH; receptor name, melanocortin type 1 receptor [MC1R]), radiolabeled alpha-MSH analogs are potential candidates for melanoma diagnosis. The aim of this study was to develop a positron emitter-labeled alpha-MSH analog suitable for PET imaging of melanoma metastases. METHODS: A short linear alpha-MSH analog, [Nle(4),Asp(5),D-Phe(7)]-alpha-MSH(4-11) (NAPamide), was newly designed and conjugated to the metal chelator DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) to enable radiometal incorporation. Compared with our previously reported DOTA-alpha-MSH analog, DOTA-MSH(oct) ([DOTA-betaAla(3),Nle(4),Asp(5),D-Phe(7),Lys(10)]-alpha-MSH(3-10)), the major modification lies in the conjugation of DOTA to the C-terminal end of the peptide via the epsilon-amino group of Lys(11), as opposed to the N-terminal alpha-amino group. After labeling with (111)In, (67)Ga, and the short-lived positron emitter (68)Ga, DOTA-NAPamide was characterized in vitro and in vivo using the mouse melanoma B16F1cell line. RESULTS: DOTA-NAPamide exhibited an almost 7-fold higher MC1R binding potency as compared with DOTA-MSH(oct). In B16F1 melanoma-bearing mice, both (111)In-DOTA-NAPamide and (67)Ga-DOTA-NAPamide behaved more favorably than (111)In-DOTA-MSH(oct). Both radiopeptides exhibited higher tumor and lower kidney uptake leading to tumor-to-kidney ratios of the 4- to 48-h area under the curve that were 4.6 times ((111)In) and 7.5 times ((67)Ga) greater than that obtained with (111)In-DOTA-MSH(oct). In addition, the 4-h kidney uptake of (67)Ga-DOTA-NAPamide could be reduced by 64% by coinjection of 15 mg L-lysine, without affecting tumor uptake. Skin primary melanoma as well as lung and liver melanoma metastases could be easily visualized on tissue section autoradiographs after systemic injection of (67)Ga-DOTA-NAPamide. The melanoma selectivity of DOTA-NAPamide was confirmed by PET imaging studies using (68)Ga-DOTA-NAPamide. Tumor uptake was found to be highest when the smallest amount of peptide was administered. CONCLUSION: DOTA-NAPamide labeled with either (111)In or (67)Ga/(68)Ga is in every way superior to (111)In-DOTA-MSH(oct) in murine models of primary and metastatic melanoma, which makes it a promising agent for melanoma targeting. High-contrast images obtained in PET studies with an experimental tumor model 1 h after injection augurs well for its clinical potential as an imaging tool.     

A (99m)Tc(CO)(3)-labeled pyrazolyl-alpha-melanocyte-stimulating hormone analog conjugate for melanoma targeting

Melanoma primary tumors can be, in most cases, removed surgically, whereas there is no satisfactory treatment for metastatic melanoma, being almost always lethal at this stage. Therefore, early detection of primary melanoma tumors is essential. The finding that melanocortin-1 receptor (MC1R) is overexpressed in isolated melanoma cells and melanoma tissues led to the radiolabeling of several alpha-melanocyte-stimulating hormone (alpha-MSH) analogs for early detection and treatment of melanoma. We have coupled the alpha-MSH analog Ac-Nle-Asp-His-d-Phe-Arg-Trp-Gly-Lys-NH(2), through the epsilon-amino group of Lys(11), to a pyrazolyl-containing chelator (pz). The resulting pz-alpha-MSH analog reacted with the fac-[(99m)Tc(CO)(3)](+) moiety, giving [Ac-Nle(4),Asp(5),d-Phe(7),Lys(11)(pz-(99m)Tc(CO)(3))]alpha-MSH(4-11) in high yield, high specific activity and high radiochemical purity. This radioconjugate, which presents remarkable stability in vitro, exhibited time- and temperature-dependent internalization (4 h at 37 degrees C; 56.7% maximum internalization) and high cellular retention (only 38% was released from the cell after 5 h) in murine melanoma B16F1 cells. A significant tumor uptake [4.2+/-0.9%ID/g, at 4 h postinjection (p.i.)] was also obtained in melanoma-bearing C57BL6 mice. The in vivo affinity and specificity of the radioconjugate to MC1R were demonstrated by receptor-blocking studies with the potent NDP-MSH agonist (63.5% reduction in tumor uptake at 4 h p.i.).     

99mTc- and 111In-labeled alpha-melanocyte-stimulating hormone peptides as imaging probes for primary and pulmonary metastatic melanoma detection.

It is highly desired to develop new imaging probes for early detection of melanoma as early diagnosis and prompt surgical removal are a patient's best hope for a cure. The purpose of this study was to determine whether (99m)Tc- and (111)In-labeled alpha-melanocyte-stimulating hormone (alpha-MSH) peptides could be used as imaging probes for primary and metastatic melanoma using dual-modality micro-SPECT/CT detection. METHODS: [Cys(3,4,10),d-Phe(7),Arg(11)]alpha-MSH(3-13) [(Arg(11))CCMSH] and [1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid]Re(Arg(11))CCMSH [DOTA-Re(Arg(11))CCMSH] were labeled with (99m)Tc and (111)In. The pharmacokinetics of (99m)Tc-(Arg(11))CCMSH were examined in B16/F1 flank and B16/F10 pulmonary metastatic murine melanoma-bearing C57 mice. The biodistribution of (111)In-DOTA-Re(Arg(11))CCMSH was performed in B16/F10 pulmonary metastatic murine melanoma-bearing C57 mice. SPECT/CT of (99m)Tc-(Arg(11))CCMSH and (111)In-DOTA-Re(Arg(11))CCMSH was determined in B16/F1 flank and B16/F10 pulmonary metastatic murine melanoma-bearing C57 mice. RESULTS: (99m)Tc-(Arg(11))CCMSH and (111)In-DOTA-Re(Arg(11))CCMSH exhibited high tumor uptakes (14.03 +/- 2.58 percentage injected dose/gram [%ID/g] at 1 h after injection and 17.29 +/- 2.49 %ID/g at 2 h after injection) in B16/F1 melanoma-bearing mice, and the flank melanoma tumors were clearly imaged by micro-SPECT/CT. Nontarget organ uptakes were considerably lower except for the kidneys. B16/F10 pulmonary melanoma metastases were also clearly visualized by micro-SPECT/CT using (99m)Tc-(Arg(11))CCMSH or (111)In-DOTA-Re(Arg(11))CCMSH as the imaging probe. (99m)Tc-(Arg(11))CCMSH exhibited images with greater resolution of metastatic melanoma lesions compared with (111)In-DOTA-Re(Arg(11))CCMSH. CONCLUSION: The favorable tumor imaging properties of (99m)Tc-(Arg(11))CCMSH and (111)In-DOTA-Re(Arg(11))CCMSH highlighted their potential as novel probes for primary and metastatic melanoma detection.     

203Pb-labeled alpha-melanocyte-stimulating hormone peptide as an imaging probe for melanoma detection.

Peptide-targeted alpha-therapy with 7.4 MBq of (212)Pb-[1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid]-ReO-[Cys(3,4,10),d-Phe(7),Arg(11)]alpha-MSH(3-13) ((212)Pb-DOTA-Re(Arg(11))CCMSH) cured 45% of B16/F1 murine melanoma-bearing C57 mice in a 120-d study, highlighting its melanoma treatment potential. However, there is a need to develop an imaging surrogate for patient-specific dosimetry and to monitor the tumor response to (212)Pb-DOTA-Re(Arg(11))CCMSH therapy. The purpose of this study was to evaluate the potential of (203)Pb-DOTA-Re(Arg(11))CCMSH as a matched-pair SPECT agent for (212)Pb-DOTA-Re(Arg(11))CCMSH. METHODS: DOTA-Re(Arg(11))CCMSH was labeled with (203)Pb in 0.5 M NH(4)OAc buffer at pH 5.4. The internalization and efflux of (203)Pb-DOTA-Re(Arg(11))CCMSH were determined in B16/F1 melanoma cells. The pharmacokinetics of (203)Pb-DOTA-Re(Arg(11))CCMSH was examined in B16/F1 melanoma-bearing C57 mice. A micro-SPECT/CT study was performed with (203)Pb-DOTA-Re(Arg(11))CCMSH in a B16/F1 melanoma-bearing C57 mouse at 2 h after injection. RESULTS: (203)Pb-DOTA-Re(Arg(11))CCMSH was easily prepared in NH(4)OAc buffer and completely separated from the excess nonradiolabeled peptide by reversed-phase high-performance liquid chromatography (RP-HPLC). (203)Pb-DOTA-Re(Arg(11))CCMSH displayed fast internalization and extended retention in B16/F1 cells. Approximately 73% of (203)Pb-DOTA-Re(Arg(11))CCMSH activity internalized after a 20-min incubation at 25 degrees C. After incubation of the cells in culture medium for 20 min, 78% of internalized activity remained in the cells. (203)Pb-DOTA-Re(Arg(11))CCMSH exhibited a biodistribution pattern similar to that of (212)Pb-DOTA-Re(Arg(11))CCMSH in B16/F1 melanoma-bearing mice. (203)Pb-DOTA-Re(Arg(11))CCMSH exhibited a peak tumor uptake of 12.00+/-3.20 percentage injected dose per gram (%ID/g) at 1 h after injection. The tumor uptake gradually decreased to 3.43+/-1.12 %ID/g at 48 h after injection. (203)Pb-DOTA-Re(Arg(11))CCMSH exhibited a peak tumor-to-kidney uptake ratio of 1.53 at 2 h after injection. The absorbed doses to the tumor and kidneys were 4.32 and 4.35 Gy, respectively, per 37 MBq. Whole-body clearance of (203)Pb-DOTA-Re(Arg(11))CCMSH was fast, with approximately 89% of the injected activity cleared through the urinary system by 2 h after injection. (203)Pb showed 1.6-mm SPECT resolution, which was comparable to (99m)Tc. Melanoma lesions were visualized through SPECT/CT images of (203)Pb-DOTA-Re(Arg(11))CCMSH at 2 h after injection. CONCLUSION: (203)Pb-DOTA-Re(Arg(11))CCMSH exhibited favorable pharmacokinetic and tumor imaging properties, highlighting its potential as a matched-pair SPECT agent for (212)Pb-DOTA-Re(Arg(11))CCMSH melanoma treatment.     

Small-animal PET of melanocortin 1 receptor expression using a 18F-labeled alpha-melanocyte-stimulating hormone analog.

(18)F-Labeled small synthetic peptides have emerged as attractive probes for imaging various molecular targets with PET. The alpha-melanocyte-stimulating hormone (alpha-MSH) receptor (melanocortin type 1 receptor [MC1R]) is overexpressed in most murine and human melanomas. It is a promising molecular target for diagnosis and therapy of melanomas. However, (18)F compounds have not been successfully developed for imaging the MC1R. METHODS: In this study, an alpha-MSH analog, Ac-Nle-Asp-His-D-Phe-Arg-Trp-Gly-Lys-NH(2) (NAPamide), was radiolabeled with N-succinimidyl-4-(18)F-fluorobenzoate ((18)F-SFB). The resulting radiopeptide was evaluated as a potential molecular probe for small-animal PET of melanoma and MC1R expression in melanoma xenografted mouse models. RESULTS: The binding affinity of (19)F-SFB-conjugated NAPamide, (19)F-FB-NAPamide, was determined to be 7.2 +/- 1.2 nM (mean +/- SD) using B16/F10 cells and (125)I-(Tyr(2))-[Nle(4),D-Phe(7)]-alpha-MSH [(125)I-(Tyr(2))-NDP] as a radioligand. The biodistribution of (18)F-FB-NAPamide was then investigated in C57BL/6 mice bearing subcutaneous murine B16/F10 melanoma tumors with high expression of MC1Rs and Fox Chase Scid mice bearing human A375M melanoma with a relatively low number of MC1R receptors. Biodistribution experiments showed that tumor uptake values (percentage injected dose per gram of tumor [%ID/g]) of (18)F-FB-NAPamide were 1.19 +/- 0.11 %ID/g and 0.46 +/- 0.11 %ID/g, in B16/F10 and A375M xenografted melanoma at 1 h after injection, respectively. Furthermore, the B16/F10 tumor uptake was significantly inhibited by coinjection with excess alpha-MSH peptide (P < 0.05), indicating that (18)F-FB-NAPamide specifically recognizes the MC1R in living mice. Small-animal PET of (18)F-FB-NAPamide in mice bearing B16/F10 and A375M tumors at 1 h after tail vein injection revealed good B16/F10 tumor-to-background contrast and low A375M tumor-to-background ratios. CONCLUSION: (18)F-FB-NAPamide is a promising molecular probe for alpha-MSH receptor-positive melanoma PET and warrants further study.     

In vivo evaluation of 99mTc/188Re-labeled linear alpha-melanocyte stimulating hormone analogs for specific melanoma targeting

Radiolabeled alpha-melanocyte stimulating hormone (alpha-MSH) analogs were examined in melanoma-bearing mice to determine the effects of peptide length, structure, and radiometal chelation chemistry on tumor targeting and in vivo biodistribution. The linear alpha-MSH analogs [Nle4,D-Phe7]alpha-MSH (NDPMSH) and [D-Phe7]alpha-MSH(5-10) (DPMSH) were radiolabeled with 99mTc and 188Re via the addition of tetrafluorophenyl mercapto-acetylglycylglycyl-gamma-aminobutyrate (MAG2) or tetrapeptide Ac-Cys-Gly-Cys-Gly (CGCG) chelation moieties. 125I-Tyr2-NDPMSH was obtained by direct iodination of the Tyr2 residue. Tumor uptake of 99mTc-labeled CGCG- and MAG2-NDPMSH analogs at 30 min postinjection were 6.52 +/- 1.11 %ID/g and 4.17 +/- 1.34 %ID/g, respectively, resulting in a significantly higher tumor-to-blood uptake ratio than that of 125I-NDPMSH or a shorter alpha-MSH analog, 99mTc-CGCG-DPMSH. The combination of radiolabeling efficacy and in vivo tumor uptake highlights the potential of 99mTc-CGCG-NDPMSH as a melanoma imaging agent.     

In vitro and in vivo investigation of matrix metalloproteinase expression in metastatic tumor models

INTRODUCTION: Overexpression of matrix metalloproteinases (MMPs), particularly MMP-2 and MMP-9, has been correlated with poor prognosis in several cancer types including lung, colon and breast. Noninvasive detection of MMP expression might allow physicians to better determine when more aggressive cancer therapy is appropriate. The peptide CTT (CTTHWGFTLC) was identified as a selective inhibitor of MMP-2/9 that inhibits the growth of MDA-MB-435 human breast cancer xenografts. METHODS: CTT was conjugated with the bifunctional chelator DOTA (1,4,7,10-tetraazacyclotetradecane-N,N',N',N'-tetraacetic acid) for radiolabeling with (64)Cu (t(1/2)=12.7 h, 17.4% beta(+), 39% beta(-)), a radionuclide suitable for positron emission tomography (PET). In vitro affinity was determined in a fluorogenic substrate assay. Tumor gelatinase targeting was evaluated in both biodistribution and microPET imaging studies. RESULTS: Cu(II)-DOTA-CTT inhibited hMMP-2 (EC(50)=8.7 microM) and mMMP-9 (EC(50)=18.2 microM) with similar affinity to CTT (hMMP-2 EC(50)=13.2 microM; mMMP-9 EC(50)=11.0 microM). In biodistribution and microPET imaging studies, (64)Cu-DOTA-CTT was taken up by MMP-2/9-positive B16F10 murine melanoma tumors. Subsequently, imaging studies using (64)Cu-DOTA-CTT were performed on MDA-MB-435 tumor-bearing mice. With zymography, tumor MMP-2/9 expression in this model was shown to be inconsistent, resulting in microPET detection of the MDA-MB-435 tumor in only 1 of 24 imaged mice. Following limited imaging success, (64)Cu-DOTA-CTT was shown to have poor in vivo stability. CONCLUSIONS: Despite some evidence for selective uptake of (64)Cu-DOTA-CTT by gelatinase-expressing tumors, the low affinity for MMP-2 and MMP-9 and in vivo instability make this an inadequate radioligand for in vivo tumor evaluation.     

Tumor-targeting properties of 90Y- and 177Lu-labeled alpha-melanocyte stimulating hormone peptide analogues in a murine melanoma model

The purpose of this study was to compare the tumor-targeting properties of (90)Y-DOTA-Re(Arg(11))CCMSH and (177)Lu-DOTA-Re(Arg(11))CCMSH in a murine melanoma mouse model. METHODS: The in vitro properties of cellular internalization and retention of (90)Y-DOTA-Re(Arg(11))CCMSH and (177)Lu-DOTA-Re(Arg(11))CCMSH were studied in B16/F1 murine melanoma cells. The pharmacokinetics of (90)Y-DOTA-Re(Arg(11))CCMSH and (177)Lu-DOTA-Re(Arg(11))CCMSH were determined in B16/F1 melanoma-bearing C57 mice. RESULTS: (90)Y-DOTA-Re(Arg(11))CCMSH and (177)Lu-DOTA-Re(Arg(11))CCMSH exhibited fast cellular internalization and extended cellular retention in B16/F1 cells. High receptor-mediated tumor uptake and retention coupled with fast whole-body clearance of (90)Y-DOTA-Re(Arg(11))CCMSH and (177)Lu-DOTA-Re(Arg(11))CCMSH were demonstrated in B16/F1 tumor-bearing C57 mice. The tumor uptakes of (90)Y-DOTA-Re(Arg(11))CCMSH and (177)Lu-DOTA-Re(Arg(11))CCMSH were 25.70 +/- 4.64 and 14.48 +/- 0.85 %ID/g at 2 h, and 14.09 +/- 2.73 and 17.68 +/- 3.32 %ID/g at 4 h postinjection. There was little activity accumulated in normal organs except for kidney. CONCLUSIONS: High tumor-targeting properties of (90)Y-DOTA-Re(Arg(11))CCMSH and (177)Lu-DOTA-Re(Arg(11))CCMSH highlighted their potential as radiopharmaceuticals for targeted radionuclide therapy of melanoma in further investigations.     

PET of vascular endothelial growth factor receptor expression.

For solid tumors and metastatic lesions, tumor vascularity is a critical factor in assessing response to therapy. Here we report the first example, to our knowledge, of (64)Cu-labeled vascular endothelial growth factor 121 (VEGF(121)) for PET of VEGF receptor (VEGFR) expression in vivo. METHODS: VEGF(121) was conjugated with 1,4,7,10-tetraazadodecane-N,N',N',N'-tetraacetic acid (DOTA) and then labeled with (64)Cu for small-animal PET of mice bearing different sized U87MG human glioblastoma xenografts. Blocking experiments and ex vivo histopathology were performed to confirm the in vivo results. RESULTS: There were 4.3 +/- 0.2 DOTA molecules per VEGF(121), and the VEGFR2 binding affinity of DOTA-VEGF(121) was comparable to VEGF(121). (64)Cu labeling of DOTA-VEGF(121) was achieved in 90 +/- 10 min and the radiolabeling yield was 87.4% +/- 3.2%. The specific activity of (64)Cu-DOTA-VEGF(121) was 3.2 +/- 0.1 GBq/mg with a radiochemical purity of >98%. Small-animal PET revealed rapid, specific, and prominent uptake of (64)Cu-DOTA-VEGF(121) in small U87MG tumors (high VEGFR2 expression) but significantly lower and sporadic uptake in large U87MG tumors (low VEGFR2 expression). No appreciable renal clearance of (64)Cu-DOTA-VEGF(121) was observed, although the kidney uptake was relatively high likely due to VEGFR1 expression. Blocking experiments, immunofluorescence staining, and western blot confirmed the VEGFR specificity of (64)Cu-DOTA-VEGF(121). CONCLUSION: Successful demonstration of the ability of (64)Cu-DOTA-VEGF(121) to visualize VEGFR expression in vivo may allow for clinical translation of this radiopharmaceutical for imaging tumor angiogenesis and guiding antiangiogenic treatment, especially patient selection and treatment monitoring of VEGFR-targeted cancer therapy.     

Therapeutic efficacy of a 188Re-labeled alpha-melanocyte-stimulating hormone peptide analog in murine and human melanoma-bearing mouse models.

The purpose of this study was to examine the therapeutic efficacy of (188)Re-(Arg(11))[Cys(3,4,10),d-Phe(7)]alpha-melanocyte-stimulating hormone(3-13) (CCMSH) in the B16/F1 murine melanoma- and TXM13 human melanoma-bearing mouse models. METHODS: (Arg(11))CCMSH was synthesized and labeled with (188)Re to form (188)Re-(Arg(11))CCMSH. B16/F1 melanoma-bearing mice were administrated 7.4 MBq, 22.2 MBq, and 2 x 14.8 MBq of (188)Re-(Arg(11))CCMSH via the tail vein. TXM13 melanoma-bearing mice were separately injected with 22.2 MBq, 2 x 14.8 MBq, and 37.0 MBq of (188)Re-(Arg(11))CCMSH through the tail vein. Two groups of 10 mice bearing either B16/F1 or TXM13 tumors were injected with saline as untreated controls. RESULTS: In contrast to the untreated control group, (188)Re-(Arg(11))CCMSH yielded rapid and lasting therapeutic effects in the treatment groups with either B16/F1 or TXM13 tumors. The tumor growth rate was reduced and the survival rate was prolonged in the treatment groups. Treatment with 2 x 14.8 MBq of (188)Re-(Arg(11))CCMSH significantly extended the mean life of B16/F1 tumor mice (P < 0.05), whereas the mean life of TXM13 tumor mice was significantly prolonged after treatment with 22.2-MBq and 37.0- MBq doses of (188)Re-(Arg(11))CCMSH (P < 0.05). High-dose (188)Re-(Arg(11))CCMSH produced no observed normal tissue toxicity. CONCLUSION: The therapy study results revealed that (188)Re-(Arg(11))CCMSH yielded significant therapeutic effects in both B16/F1 murine melanoma- and TXM13 human melanoma-bearing mouse models. (188)Re-(Arg(11))CCMSH appears to be a promising radiolabeled peptide for targeted radionuclide therapy of melanoma.     

Melanoma targeting with DOTA-alpha-melanocyte-stimulating hormone analogs: structural parameters affecting tumor uptake and kidney uptake.

Radiolabeled analogs of alpha-melanocyte-stimulating hormone (alpha-MSH) are potential candidates for the diagnosis and therapy of melanoma metastases. After our recent observation that a linear octapeptide alpha-MSH analog incorporating the metal chelator 1,4,7,10-tetraazacyclododecane-N,N',N',N'-tetraacetic acid (DOTA) at the C-terminal lysine, [Nle(4),Asp(5),d-Phe(7),Lys(11)(DOTA)]-alpha-MSH(4-11) (DOTA-NAPamide), showed high accumulation in melanomas in a mouse model, low uptake in normal tissues, and moderate uptake in the kidneys, we attempted to identify the structural parameters influencing tumor uptake versus kidney uptake. METHODS: We designed a series of novel DOTA-alpha-MSH analogs differing from DOTA-NAPamide by small alterations, such as the position of DOTA in the peptide, hydrophobicity, and charge, by modifying the C-terminal Lys(11) residue. They were evaluated both for their melanocortin type 1 receptor (MC1R)-binding potency and for their biodistribution by use of the B16F1 melanoma mouse model. RESULTS: When DOTA was shifted to the N terminus of the peptide, a 3-fold increase in kidney retention was obtained. However, when the epsilon-amino group of the Lys(11) residue was acetylated in addition to the DOTA relocation, kidney uptake returned to the low values obtained with DOTA-NAPamide; this result indicated that neutralization of the epsilon-amino group positive charge of the Lys(11) residue rather than the position of DOTA accounted for the low kidney retention. Unexpectedly, no further reduction in kidney uptake was obtained by the introduction of 1 or 2 negative charges on Lys(11). Melanoma uptake was in accordance with MC1R affinity; the highest values were obtained for peptides bearing carboxy-terminal amidation and positioning of DOTA. CONCLUSION: The kidney uptake of DOTA-alpha-MSH analogs could be considerably reduced, without affecting MC1R affinity, by altering (neutralizing) the charge of the Lys(11) residue. Accordingly, the resulting peptides exhibited a high ratio of tumor uptake to kidney uptake that is favorable for diagnostic and therapeutic applications. These structure-activity data may help to improve the performance of DOTA-alpha-MSH analogs and other radiopeptides.     

Reducing renal uptake of 90Y- and 177Lu-labeled alpha-melanocyte stimulating hormone peptide analogues

OBJECTIVE: The purpose of this study was to improve the tumor-to-kidney uptake ratios of (90)Y- and (177)Lu-[1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-Re-Cys(3,4,10), d-Phe(7), Arg(11)]alpha-melanocyte stimulating hormone(3-13) {DOTA-Re(Arg(11))CCMSH} through coupling a negatively charged glutamic acid (Glu) to the peptide sequence. METHODS: A new peptide of DOTA-Re(Glu(2), Arg(11))CCMSH was designed, synthesized and labeled with (90)Y and (177)Lu. Pharmacokinetics of (90)Y- and (177)Lu-DOTA-Re(Glu(2), Arg(11))CCMSH was determined in B16/F1 murine melanoma-bearing C57 mice. RESULTS: (90)Y- and (177)Lu-DOTA-Re(Glu(2), Arg(11))CCMSH exhibited significantly (P<.05) less renal uptake values than (90)Y- and (177)Lu-DOTA-Re(Arg(11))CCMSH at 30 min and at 2, 4 and 24 h after dose administration. The renal uptake values of (90)Y- and (177)Lu-DOTA-Re(Glu(2), Arg(11))CCMSH were 28.16% and 28.81% of those of (90)Y- and (177)Lu-DOTA-Re(Arg(11))CCMSH, respectively, at 4 h postinjection. (90)Y- and (177)Lu-DOTA-Re(Glu(2), Arg(11))CCMSH displayed higher tumor-to-kidney uptake ratios than (90)Y- and (177)Lu-DOTA-Re(Arg(11))CCMSH at 30 min and at 2, 4 and 24 h after dose administration. The tumor-to-kidney uptake ratio of (90)Y- and (177)Lu-DOTA-Re(Glu(2), Arg(11))CCMSH was 2.28 and 1.69 times of (90)Y- and (177)Lu-DOTA-Re(Arg(11))CCMSH, respectively, at 4 h postinjection. The (90)Y- and (177)Lu-DOTA-Re(Glu(2), Arg(11))CCMSH activity accumulation was low in normal organs except for kidney. CONCLUSIONS: Coupling a negatively charged amino acid (Glu) to the CCMSH peptide sequence dramatically reduced the renal uptake values and increased the tumor-to-kidney uptake ratios of (90)Y- and (177)Lu-DOTA-Re(Glu(2), Arg(11))CCMSH, facilitating their potential applications as radiopharmaceuticals for targeted radionuclide therapy of melanoma.     

Melanoma imaging with pretargeted bivalent bacteriophage.

Random bacteriophage (phage) display peptide libraries have traditionally been used for the selection of clones that bind specific tissues, tumors, and antigens. However, once the targeting peptide is synthetically produced, it often displays a lower affinity than the original phage because of a lack of avidity effects and removal from the virion surface. We hypothesized that multivalent bifunctional phage displaying peptides that target novel molecular biomarkers would facilitate the in vivo imaging of cancer. This study provides proof of principle for the use of phage displaying multiple melanocortin-1 receptor-homing peptides for the pretargeting and subsequent imaging of murine melanomas in vivo. METHODS: A 2-step melanoma pretargeting-imaging system was developed by first generating and biotinylating phage that displayed up to 5 copies of alpha-melanocyte-stimulating hormone (alpha-MSH) peptide analogs. Second, streptavidin was conjugated to diethylenetriaminepentaacetic acid for the purpose of radiolabeling with (111)In. RESULTS: The specificity of the MSH2.0 phage for the B16-F1 melanoma was demonstrated both in vitro and in vivo. In vitro micropanning assays with phage at inputs of 10(7) and 10(6) transducing units per milliliter resulted in approximately 200- and approximately 1,000-fold-greater recovery of the MSH2.0 phage over the background, respectively. In vivo distribution studies indicated that melanoma uptake values were 2.6 +/- 1.1, 0.6 +/- 0.2, and 1.0 +/- 0.1 (mean +/- SD) percentage injected dose per gram at 0.5, 6, and 24 h after the injection of (111)In-radiolabeled streptavidin ((111)In-SA). The accumulation of radioactivity within the tumor was 1.8 times greater for the biotinylated MSH2.0 phage than for the biotinylated wild-type phage. These data, combined with reduction by 2.4-fold through competition with a nonradiolabeled alpha-MSH peptide analog, indicated the specific targeting of melanoma tumors in vivo. SPECT/CT image analysis of B16-F1 melanoma-bearing mice showed that intravenously injected biotinylated alpha-MSH phage were retained within melanoma tumors at 4 h after injection of (111)In-SA. CONCLUSION: This study demonstrated the use of multivalent bifunctional phage in a 2-step pretargeting-imaging system.     

(64)Cu-labeled tetrameric and octameric RGD peptides for small-animal PET of tumor alpha(v)beta(3) integrin expression.

Integrin alpha(v)beta(3) plays a critical role in tumor angiogenesis and metastasis. Suitably radiolabeled cyclic arginine-glycine-aspartic (RGD) peptides can be used for noninvasive imaging of alpha(v)beta(3) expression and targeted radionuclide therapy. In this study, we developed (64)Cu-labeled multimeric RGD peptides, E{E[c(RGDyK)](2)}(2) (RGD tetramer) and E(E{E[c(RGDyK)](2)}(2))(2) (RGD octamer), for PET imaging of tumor integrin alpha(v)beta(3) expression. METHODS: Both RGD tetramer and RGD octamer were synthesized with glutamate as the linker. After conjugation with 1,4,7,10-tetra-azacyclododecane-N,N',N',N'-tetraacetic acid (DOTA), the peptides were labeled with (64)Cu for biodistribution and small-animal PET imaging studies (U87MG human glioblastoma xenograft model and c-neu oncomouse model). A cell adhesion assay, a cell-binding assay, receptor blocking experiments, and immunohistochemistry were also performed to evaluate the alpha(v)beta(3)-binding affinity/specificity of the RGD peptide-based conjugates in vitro and in vivo. RESULTS: RGD octamer had significantly higher integrin alpha(v)beta(3)-binding affinity and specificity than RGD tetramer analog (inhibitory concentration of 50% was 10 nM for octamer vs. 35 nM for tetramer). (64)Cu-DOTA-RGD octamer had higher tumor uptake and longer tumor retention than (64)Cu-DOTA-RGD tetramer in both tumor models tested. The integrin alpha(v)beta(3) specificity of both tracers was confirmed by successful receptor-blocking experiments. The high uptake and slow clearance of (64)Cu-DOTA-RGD octamer in the kidneys was attributed mainly to the integrin positivity of the kidneys, significantly higher integrin alpha(v)beta(3)-binding affinity, and the larger molecular size of the octamer, as compared with the other RGD analogs. CONCLUSION: Polyvalency has a profound effect on the receptor-binding affinity and in vivo kinetics of radiolabeled RGD multimers. The information obtained here may guide the future development of RGD peptide-based imaging and internal radiotherapeutic agents targeting integrin alpha(v)beta(3).     

PET/MRI dual-modality tumor imaging using arginine-glycine-aspartic (RGD)-conjugated radiolabeled iron oxide nanoparticles

The purpose of this study was to develop a bifunctional iron oxide (IO) nanoparticle probe for PET and MRI scans of tumor integrin alphavbeta3 expression. METHODS: Polyaspartic acid (PASP)-coated IO (PASP-IO) nanoparticles were synthesized using a coprecipitation method, and particle size and magnetic properties were measured. A phantom study was used to assess the efficacy of PASP-IO as a T2-weighted MRI contrast agent. PASP-IO nanoparticles with surface amino groups were coupled to cyclic arginine-glycine-aspartic (RGD) peptides for integrin alphavbeta3 targeting and macrocyclic 1,4,7,10-tetraazacyclododecane-N,N',N',N',-tetraacetic acid (DOTA) chelators for PET after labeling with 64Cu. IO nanoparticle conjugates were further tested in vitro and in vivo to determine receptor targeting efficacy and feasibility for dual PET/MRI. RESULTS: PASP-IO nanoparticles made by single-step reaction have a core size of 5 nm with a hydrodynamic diameter of 45 +/- 10 nm. The saturation magnetization of PASP-IO nanoparticles is about 117 emu/g of iron, and the measured r2 and r2* are 105.5 and 165.5 (s.mM)(-1), respectively. A displacement competitive binding assay indicates that DOTA-IO-RGD conjugates bound specifically to integrin alphavbeta3 in vitro. Both small-animal PET and T2-weighted MRI show integrin-specific delivery of conjugated RGD-PASP-IO nanoparticles and prominent reticuloendothelial system uptake. CONCLUSION: We have successfully developed an IO-based nanoprobe for simultaneous dual PET and MRI of tumor integrin expression. The success of this bifunctional imaging approach may allow for earlier tumor detection with a high degree of accuracy and provide further insight into the molecular mechanisms of cancer.     

Comparative in vitro and in vivo evaluation of two 64Cu-labeled bombesin analogs in a mouse model of human prostate adenocarcinoma

Bombesin (BBN), an analog of human gastrin-releasing peptide (GRP), binds to the GRP receptor (GRPR) with high affinity and specificity. Overexpression of GRPR has been discovered in mostly androgen-independent human prostate tissues and, thus, provides a potential target for prostate cancer diagnosis and therapy. We have previously demonstrated the feasibility of the positron emission tomography (PET) imaging using 64Cu-1,4,7,10-tetraazadodecane-N,N',N',N'-tetraacetic acid (DOTA)-[Lys3]BBN to detect GRPR-positive prostate cancer. In this study, we compared the receptor affinity, metabolic stability, tumor-targeting efficacy, and pharmacokinetics of a truncated BBN analog 64Cu-DOTA-Aca-BBN(7-14) with 64Cu-DOTA-[Lys3]BBN. Binding of each DOTA conjugate to GRPR on PC-3 and 22Rv1 prostate cancer cells was evaluated with competitive binding assay using 125I-[Tyr4]BBN as radioligand. In vivo pharmacokinetics was determined on male nude mice subcutaneously implanted with PC-3 cells. Dynamic microPET imaging was performed to evaluate the systemic distribution of the tracers. Metabolic stability of the tracers in blood, urine, tumor, liver and kidney was studied using high-performance liquid chromatography. The results showed that 125I-[Tyr4]BBN has a K(d) of 14.8+/-0.4 nM against PC-3 cells, and the receptor concentration on PC-3 cell surface is approximately 2.7+/-0.1 x 10(6) receptors per cell. The 50% inhibitory concentration value for DOTA-Aca-BBN(7-14) is 18.4 +/- 0.2 nM, and that for DOTA-[Lys3]BBN is 2.2 +/- 0.5 nM. DOTA-[Lys3]BBN shows a better tumor contrast and absolute tumor activity accumulation compared to DOTA-Aca-BBN(7-14). Studies on metabolic stability for both tracers on organ homogenates showed that 64Cu-DOTA-[Lys3]BBN is relatively stable. This study demonstrated that both tracers are suitable for targeted PET imaging to detect the expression of GRPR in prostate cancer, while 64Cu-DOTA-[Lys3]BBN may have a better potential for clinical translation.     

Evaluation of an (111)In-DOTA-rhenium cyclized alpha-MSH analog: a novel cyclic-peptide analog with improved tumor-targeting properties.

The aim of this study was to examine the effect of rhenium-mediated peptide cyclization on melanoma targeting, biodistribution, and clearance kinetics of the alpha-melanocyte-stimulating hormone (alpha-MSH) analog 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) coupled ReO-cyclized [Cys(3,4,10),D-Phe(7)]alpha-MSH(3-13) (DOTA-ReCCMSH). METHODS: DOTA-ReCCMSH was compared with its reduced nonmetalated linear homolog, DOTA-CCMSH, and an analog in which rhenium cyclization was replaced by disulfide bond cyclization, DOTA-[Cys(4,10),D-Phe(7)]alpha-MSH(4-13) (CMSH). DOTA was also conjugated to the amino terminus of one of the highest-affinity alpha-MSH receptor-binding peptides, [Nle(4),D-Phe(7)]alpha-MSH (NDP), as a linear peptide standard. The DOTA-conjugated alpha-MSH analogs were radiolabeled with (111)In and examined for their in vitro receptor-binding affinity with B16/F1 murine melanoma cells, and their in vivo biodistribution properties were evaluated and compared in melanoma tumor-bearing C57 mice. RESULTS: The tumor uptake values of (111)In-DOTA-ReCCMSH were significantly higher than those of the other closely related (111)In-DOTA-alpha-MSH conjugates. Even at 24 h after injection, a comparison of the tumor uptake values for (111)In-DOTA-coupled ReCCMSH (4.86 +/- 1.52 percentage injected dose [%ID]/g), CCMSH (1.91 +/- 0.56 %ID/g), CMSH (3.09 +/- 0.32 %ID/g), and NDP (2.47 +/- 0.79 %ID/g) highlighted the high tumor retention property of ReCCMSH. Rhenium-coordinated cyclization resulted in less renal radioactivity accumulation of (111)In-DOTA-ReCCMSH (8.98 +/- 0.82 %ID/g) than of (111)In-DOTA-CCMSH (63.2 +/- 15.6 %ID/g), (111)In-DOTA-CMSH (38.4 +/- 3.6 %ID/g), and (111)In-DOTA-NDP (12.0 +/- 1.96 %ID/g) at 2 h after injection and significantly increased its clearance into the urine (92 %ID at 2 h after injection). A high radioactivity uptake ratio of tumor to normal tissue was obtained for (111)In-DOTA-ReCCMSH (e.g., 489, 159, 100, and 49 for blood, muscle, lung, and liver, respectively, at 4 h after injection). CONCLUSION: The novel ReO-coordinated cyclic structure of DOTA-ReCCMSH contributes significantly to its enhanced tumor-targeting and renal clearance properties and makes DOTAReCCMSH an excellent candidate for melanoma radiodetection and radiotherapy.     

Pegylated Arg-Gly-Asp peptide: 64Cu labeling and PET imaging of brain tumor alphavbeta3-integrin expression.

The alphav-integrins, cell adhesion molecules that are highly expressed on activated endothelial cells and tumor cells but not on dormant endothelial cells or normal cells, present an attractive target for tumor imaging and therapy. We previously coupled a cyclic Arg-Gly-Asp (RGD) peptide, c(RGDyK), with 1,4,7,10-tetraazacyclododecane-N,N',N',N'-tetraacetic acid (DOTA) and labeled the RGD-DOTA conjugate with 64Cu (half-life, 12.8 h; 19% beta+) for solid tumor targeting, with high tumor-to-background contrast. The rapid tumor washout rate and persistent liver and kidney retention of this tracer prompted us to optimize the tracer for improved pharmacokinetic behavior. In this study, we introduced a polyethylene glycol (PEG; molecular weight, 3,400) moiety between DOTA and RGD and evaluated the 64Cu-DOTA-PEG-RGD tracer for microPET imaging in brain tumor models. METHODS: DOTA was activated in situ and conjugated with RGD-PEG-NH2 under slightly basic conditions. alphavbeta3-Integrin-binding affinity was evaluated with a solid-phase receptor-binding assay in the presence of 125I-echistatin. Female nude mice bearing subcutaneous U87MG glioblastoma xenografts were administered 64Cu-DOTA-PEG-RGD, and the biodistributions of the radiotracer were evaluated from 30 min to 4 h after injection. microPET (20 min of static imaging at 1 h after injection) and then quantitative autoradiography were used for tumor visualization and quantification. The same tracer was also applied to an orthotopic U87MG model for tumor detection. RESULTS: The radiotracer was synthesized with a high specific activity (14,800-29,600 GBq/mmol [400-800 Ci/mmol]). The c(RGDyK)-PEG-DOTA ligand showed intermediate binding affinity for alphavbeta3-integrin (50% inhibitory concentration, 67.5 +/- 7.8 nmol/L [mean +/- SD]). The pegylated RGD peptide demonstrated rapid blood clearance (0.57 +/- 0.15 percentage injected dose [%ID]/g [mean +/- SD] at 30 min after injection and 0.03 +/- 0.02 %ID/g at 4 h after injection). Activity accumulation in the tumor was rapid and high at early time points (2.74 +/- 0.45 %ID/g at 30 min after injection), and some activity washout was seen over time (1.62 +/- 0.18 %ID/g at 4 h after injection). Compared with (64)Cu-DOTA-RGD, this tracer showed improved in vivo kinetics, with significantly reduced liver uptake (0.99 +/- 0.08 %ID/g vs. 1.73 +/- 0.39 %ID/g at 30 min after injection and 0.58 +/- 0.07 %ID/g vs. 2.57 +/- 0.49 %ID/g at 4 h after injection). The pegylated RGD peptide showed higher renal accumulation at early time points (3.51 +/- 0.24 %ID/g vs. 2.18 +/- 0.23 %ID/g at 30 min after infection) but more rapid clearance (1.82 +/- 0.29 %ID/g vs. 2.01 +/- 0.25 %ID/g at 1 h after injection) than 64Cu-DOTA-RGD. The integrin receptor specificity of this radiotracer was demonstrated by blocking of tumor uptake by coinjection with nonradiolabeled c(RGDyK). The high tumor-to-organ ratios for the pegylated RGD peptide tracer (at 1 h after injection: tumor-to-blood ratio, 20; tumor-to-muscle ratio, 12; tumor-to-liver ratio, 2.7; and tumor-to-kidney ratio, 1.2) were confirmed by microPET and autoradiographic imaging in a subcutaneous U87MG tumor model. This tracer was also able to detect an orthotopic brain tumor in a model in which U87MG cells were implanted into the mouse forebrain. Although the magnitude of tumor uptake in the orthotopic xenograft was lower than that in the subcutaneous xenograft, the orthotopic tumor was still visualized with clear contrast from normal brain tissue. CONCLUSION: This study demonstrated the suitability of a PEG moiety for improving the in vivo kinetics of a 64Cu-RGD peptide tracer without compromising the tumor-targeting ability and specificity of the peptide. Systematic investigations of the effects of the size and geometry of PEG on tumor targeting and in vivo kinetics will lead to the development of radiotracers suitable for clinical applications such as visualizing and quantifying alphav-integrin expression by PET. In addition, the same ligand labeled with therapeutic radionuclides may be applicable for integrin-targeted internal radiotherapy.