Effect of Ibuprofen as an Albumin Binder on Melanoma-Targeting Properties of 177Lu-Labeled Ibuprofen-Conjugated Alpha-Melanocyte-Stimulating Hormone Peptides.

The purpose of this study was to examine how the introduction of ibuprofen (IBU) affected tumor-targeting and biodistribution properties of 177Lu-labeled IBU-conjugated alpha-melanocyte-stimulating hormone peptides. The IBU was used as an albumin binder and conjugated to the DOTA-Lys moiety without or with a linker to yield DOTA-Lys(IBU)-GG-Nle-CycMSHhex {1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-Lys(IBU)-Gly-Gly-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2}, DOTA-Lys(Asp-IBU)-GGNle-CycMSHhex, DOTA-Lys(Asn-IBU)-GGNle-CycMSHhex, and DOTA-Lys(Dab-IBU)-GGNle-CycMSHhex peptides. Their melanocortin-receptor 1 (MC1R) binding affinities were determined on B16/F10 melanoma cells first. Then the biodistribution of 177Lu-labeled peptides was determined on B16/F10 melanoma-bearing C57 mice at 2 h postinjection to choose the lead peptide for further examination. The full biodistribution and melanoma imaging properties of 177Lu-DOTA-Lys(Asp-IBU)-GGNle-CycMSHhex were further evaluated using B16/F10 melanoma-bearing C57 mice. DOTA-Lys(IBU)-GG-Nle-CycMSHhex, DOTA-Lys(Asp-IBU)-GGNle-CycMSHhex, DOTA-Lys(Asn-IBU)-GGNle-CycMSHhex, and DOTA-Lys(Dab-IBU)-GGNle-CycMSHhex displayed the IC50 values of 1.41 ± 0.37, 1.52 ± 0.08, 0.03 ± 0.01, and 0.58 ± 0.06 nM on B16/F10 melanoma cells, respectively. 177Lu-DOTA-Lys(Asp-IBU)-GGNle-CycMSHhex exhibited the lowest liver and kidney uptake among all four designed 177Lu peptides. Therefore, 177Lu-DOTA-Lys(Asp-IBU)-GGNle-CycMSHhex was further evaluated for its full biodistribution and melanoma imaging properties. The B16/F10 melanoma uptake of 177Lu-DOTA-Lys(Asp-IBU)-GGNle-CycMSHhex was 19.5 ± 3.12, 24.12 ± 3.35, 23.85 ± 2.08, and 10.80 ± 2.89% ID/g at 0.5, 2, 4, and 24 h postinjection, respectively. Moreover, 177Lu-DOTA-Lys(Asp-IBU)-GGNle-CycMSHhex could clearly visualize the B16/F10 melanoma lesions at 2 h postinjection. The conjugation of IBU with or without a linker to GGNle-CycMSHhex affected the MC1R binding affinities of the designed peptides. The charge of the linker played a key role in the liver and kidney uptake of 177Lu-Asp-IBU, 177Lu-Asn-IBU, and 177Lu-Dab-IBU. 177Lu-Asp-IBU exhibited higher tumor/liver and tumor/kidney uptake ratios than those of 177Lu-Asn-IBU and 177Lu-Dab-IBU, underscoring its potential evaluation for melanoma therapy in the future.

Elevating theranostics: The emergence and promise of radiopharmaceutical cell-targeting heterodimers in human cancers.

Optimal therapeutic and diagnostic efficacy is essential for healthcare's global mission of advancing oncologic drug development. Accurate diagnosis and detection are crucial prerequisites for effective risk stratification and personalized patient care in clinical oncology. A paradigm shift is emerging with the promise of multi-receptor-targeting compounds. While existing detection and staging methods have demonstrated some success, the traditional approach of monotherapy is being reevaluated to enhance therapeutic effectiveness. Heterodimeric site-specific agents are a versatile solution by targeting two distinct biomarkers with a single theranostic agent. This review describes the innovation of dual-targeting compounds, examining their design strategies, therapeutic implications, and the promising path they present for addressing complex diseases.

Brown adipose tissue metabolism in women is dependent on ovarian status.

In rodents, loss of estradiol (E2) reduces brown adipose tissue (BAT) metabolic activity. Whether E2 impacts BAT activity in women is not known. BAT oxidative metabolism was measured in premenopausal (n = 27; 35 ± 9 yr; body mass index = 26.0 ± 5.3 kg/m2) and postmenopausal (n = 25; 51 ± 8 yr; body mass index = 28.0 ± 5.0 kg/m2) women at room temperature and during acute cold exposure using [11C]acetate with positron emission tomography coupled with computed tomograph. BAT glucose uptake was also measured during acute cold exposure using 2-deoxy-2-[18F]fluoro-d-glucose. To isolate the effects of ovarian hormones from biological aging, measurements were repeated in a subset of premenopausal women (n = 8; 40 ± 4 yr; BMI = 28.0 ± 7.2 kg/m2) after 6 mo of gonadotropin-releasing hormone agonist therapy to suppress ovarian hormones. At room temperature, there was no difference in BAT oxidative metabolism between premenopausal (0.56 ± 0.31 min-1) and postmenopausal women (0.63 ± 0.28 min-1). During cold exposure, BAT oxidative metabolism (1.28 ± 0.85 vs. 0.91 ± 0.63 min-1, P = 0.03) and net BAT glucose uptake (84.4 ± 82.5 vs. 29.7 ± 31.4 nmol·g-1·min-1, P < 0.01) were higher in premenopausal than postmenopausal women. In premenopausal women who underwent gonadotropin-releasing hormone agonist, cold-stimulated BAT oxidative metabolism was reduced to a similar level (from 1.36 ± 0.66 min-1 to 0.91 ± 0.41 min-1) to that observed in postmenopausal women (0.91 ± 0.63 min-1). These results provide the first evidence in humans that reproductive hormones are associated with BAT oxidative metabolism and suggest that BAT may be a target to attenuate age-related reduction in energy expenditure and maintain metabolic health in postmenopausal women.NEW & NOTEWORTHY In rodents, loss of estrogen reduces brown adipose tissue (BAT) activity. Whether this is true in humans is not known. We found that BAT oxidative metabolism and glucose uptake were lower in postmenopausal compared to premenopausal women. In premenopausal women who underwent ovarian suppression to reduce circulating estrogen, BAT oxidative metabolism was reduced to postmenopausal levels. Thus the loss of ovarian function in women leads to a reduction in BAT metabolic activity independent of age.

Introduction of a Polyethylene Glycol Linker Improves Uptake of 67Cu-NOTA-Conjugated Lactam-Cyclized Alpha-Melanocyte-Stimulating Hormone Peptide in Melanoma.

The aim of this study was to evaluate the effect of linker on tumor targeting and biodistribution of 67Cu-NOTA-PEG2Nle-CycMSHhex {67Cu-1,4,7-triazacyclononane-1,4,7-triyl-triacetic acid-polyethylene glycol-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} and 67Cu-NOTA-GGNle-CycMSHhex {67Cu-NOTA-GlyGlyNle-CycMSHhex} on melanoma-bearing mice. NOTA-PEG2Nle-CycMSHhex and NOTA-GGNle-CycMSHhex were synthesized and purified by HPLC. The biodistribution of 67Cu-NOTA-PEG2Nle-CycMSHhex and 67Cu-NOTA-GGNle-CycMSHhex was determined in B16/F10 melanoma-bearing C57 mice. The melanoma imaging property of 67Cu-NOTA-PEG2Nle-CycMSHhex was further examined in B16/F10 melanoma-bearing C57 mice. 67Cu-NOTA-PEG2Nle-CycMSHhex exhibited higher tumor uptake than 67Cu-NOTA-GGNle-CycMSHhex at 2, 4, and 24 h post-injection. The tumor uptake of 67Cu-NOTA-PEG2Nle-CycMSHhex was 27.97 ± 1.98, 24.10 ± 1.83, and 9.13 ± 1.66% ID/g at 2, 4, and 24 h post-injection, respectively. Normal organ uptake of 67Cu-NOTA-PEG2Nle-CycMSHhex was lower than 2.6% ID/g at 4 h post-injection, except for kidney uptake. The renal uptake of 67Cu-NOTA-PEG2Nle-CycMSHhex was 6.43 ± 1.31, 2.60 ± 0.79, and 0.90 ± 0.18% ID/g at 2, 4, and 24 h post-injection, respectively. 67Cu-NOTA-PEG2Nle-CycMSHhex showed high tumor to normal organ uptake ratios after 2 h post-injection. The B16/F10 melanoma lesions could be clearly visualized by single photon emission computed tomography (SPECT) using 67Cu-NOTA-PEG2Nle-CycMSHhex as an imaging probe at 4 h post-injection. The favorable tumor targeting and biodistribution properties of 67Cu-NOTA-PEG2Nle-CycMSHhex underscored its potential as an MC1R-targeted therapeutic peptide for melanoma treatment.

Effects of Polyethylene Glycol and 8-Aminooctanoic Acid Linkers on Melanoma Uptake of [99mTc]Tc-Tricarbonyl-NOTA-Conjugated Lactam-Cyclized α-MSH Peptides.

The purpose of this study was to evaluate the effect of linkers on tumor targeting and biodistribution of [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex {[99mTc]Tc(CO)3-1,4,7-triazacyclononane-1,4,7-triyl-triacetic acid-polyethylene glycol-Nle-c[Asp-His-d-Phe-Arg-Trp-Lys]-CONH2} and [99mTc]Tc(CO)3-NOTA-AocNle-CycMSHhex {[99mTc]Tc(CO)3-NOTA-8-aminooctanoic acid-Nle-CycMSHhex} on B16/F10 melanoma-bearing mice. NOTA-PEG2Nle-CycMSHhex and NOTA-AocNle-CycMSHhex were synthesized and radiolabeled with [99mTc]Tc via the {[99mTc]Tc(CO)3(OH2)3}+ intermediate. The biodistribution of [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex and [99mTc]Tc(CO)3-NOTA-AocNle-CycMSHhex was determined on B16/F10 melanoma-bearing C57 mice. The melanoma imaging property of [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex was determined on B16/F10 melanoma-bearing C57 mice. [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex and [99mTc]Tc(CO)3-NOTA-AocNle-CycMSHhex were readily prepared with more than 90% radiochemical yields and exhibited MC1R-specific binding on B16/F10 melanoma cells. [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex exhibited a higher tumor uptake than [99mTc]Tc(CO)3-NOTA-AocNle-CycMSHhex at 2, 4, and 24 h postinjection. The tumor uptake of [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex was 13.63 ± 1.13, 31.93 ± 2.57, 20.31 ± 3.23, and 1.33 ± 0.15% ID/g at 0.5, 2, 4, and 24 h postinjection, respectively. The tumor uptake of [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex was 1.6 and 3.4 times the tumor uptake of [99mTc]Tc(CO)3-NOTA-AocNle-CycMSHhex at 2 and 4 h postinjection, respectively. Meanwhile, the normal organ uptake of [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex was lower than 1.8% ID/g at 2 h postinjection. The renal uptake of [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex was only 1.73 ± 0.37, 0.73 ± 0.14, and 0.03 ± 0.01% ID/g at 2, 4, and 24 h postinjection, respectively. [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex showed high tumor to normal organ uptake ratios at 2 h postinjection. Single-photon emission computed tomography imaging revealed that the B16/F10 melanoma lesions could be clearly visualized by [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex at 2 h postinjection. Overall, the high tumor uptake and low kidney uptake of [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex highlighted its potential for melanoma imaging and warranted the future evaluation of [188Re]Re(CO)3-NOTA-PEG2Nle-CycMSHhex for melanoma therapy.

Novel Al18F-NOTA-Conjugated Lactam-Cyclized α-Melanocyte-Stimulating Hormone Peptides with Enhanced Melanoma Uptake.

The purpose of this study was to evaluate the effect of linker on tumor targeting and biodistribution of Al18F-NOTA-PEG2Nle-CycMSHhex {Al18F-1,4,7-triazacyclononane-1,4,7-triyl-triacetic acid-poly(ethylene glycol)-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} and Al18F-NOTA-AocNle-CycMSHhex {Al18F-NOTA-8-aminooctanoic acid-Nle-CycMSHhex} on melanoma-bearing mice. NOTA-PEG2Nle-CycMSHhex and NOTA-AocNle-CycMSHhex were synthesized using fluorenylmethoxycarbonyl (Fmoc) chemistry. The melanocortin-1 (MC1) receptor binding affinities of the peptides were determined on B16/F10 melanoma cells. The biodistribution of Al18F-NOTA-PEG2Nle-CycMSHhex and Al18F-NOTA-AocNle-CycMSHhex was determined on B16/F10 melanoma-bearing C57 mice. The melanoma imaging property of Al18F-NOTA-PEG2Nle-CycMSHhex was further examined on B16/F10 melanoma-bearing C57 mice because of its higher melanoma uptake and lower renal uptake than that of Al18F-NOTA-AocNle-CycMSHhex. The IC50 values of NOTA-PEG2/AocNle-CycMSHhex were 1.24 ± 0.07 and 2.75 ± 0.48 nM on B10/F10 cells. Al18F-NOTA-PEG2Nle-CycMSHhex and Al18F-NOTA-AocNle-CycMSHhex were readily prepared with more than 55% of radiolabeling yields and displayed melanocortin-1 receptor (MC1R)-specific binding on B16/F10 cells. Al18F-NOTA-PEG2Nle-CycMSHhex exhibited higher tumor uptake and lower kidney and liver uptake than Al18F-NOTA-AocNle-CycMSHhex at 1 and 2 h post injection. The tumor and renal uptakes of Al18F-NOTA-PEG2Nle-CycMSHhex were 17.44 ± 0.76 and 2.07 ± 0.43% ID/g at 1 h post injection, respectively. Al18F-NOTA-PEG2Nle-CycMSHhex showed the high tumor to normal organ uptake ratios after 1 h post injection. The B16/F10 melanoma lesions could be clearly visualized by positron emission tomography (PET) using Al18F-NOTA-PEG2Nle-CycMSHhex as an imaging probe at 1 and 2 h post injection. Overall, high tumor uptake, low kidney and liver uptake, and fast urinary clearance of Al18F-NOTA-PEG2Nle-CycMSHhex highlighted its potential as an MC1R-targeted imaging probe for melanoma detection.

Highlight selection of radiochemistry and radiopharmacy developments by editorial board.

BACKGROUND: The Editorial Board of EJNMMI Radiopharmacy and Chemistry releases a biyearly highlight commentary to update the readership on trends in the field of radiopharmaceutical development. RESULTS: This commentary of highlights has resulted in 23 different topics selected by each member of the Editorial Board addressing a variety of aspects ranging from novel radiochemistry to first in man application of novel radiopharmaceuticals and also a contribution in relation to MRI-agents is included. CONCLUSION: Trends in (radio)chemistry and radiopharmacy are highlighted demonstrating the progress in the research field being the scope of EJNMMI Radiopharmacy and Chemistry.

Novel 64Cu-Labeled NOTA-Conjugated Lactam-Cyclized Alpha-Melanocyte-Stimulating Hormone Peptides with Enhanced Tumor to Kidney Uptake Ratios.

The aim of this study was to evaluate the effect of linker on tumor targeting and biodistribution of 64Cu-NOTA-PEG2Nle-CycMSHhex {64Cu-1,4,7-triazacyclononane-1,4,7-triyl-triacetic acid-polyethylene glycol-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} and 64Cu-NOTA-AocNle-CycMSHhex {64Cu-NOTA-8-aminooctanoic acid-Nle-CycMSHhex} on melanoma-bearing mice. NOTA-PEG2Nle-CycMSHhex and NOTA-AocNle-CycMSHhex were synthesized and purified by HPLC. The melanocortin-1 (MC1) receptor binding affinities of the peptides were examined on B16/F10 melanoma cells. The biodistributions of 64Cu-NOTA-PEG2Nle-CycMSHhex and 64Cu-NOTA-AocNle-CycMSHhex were determined on B16/F10 melanoma-bearing C57 mice. The melanoma imaging property of 64Cu-NOTA-PEG2Nle-CycMSHhex was further examined on B16/F10 melanoma-bearing C57 mice because of its higher melanoma uptake than 64Cu-NOTA-AocNle-CycMSHhex. The IC50 values of NOTA-PEG2Nle-CycMSHhex and NOTA-AocNle-CycMSHhex were 1.24 ± 0.07 and 2.75 ± 0.48 nM on B10/F10 melanoma cells. 64Cu-NOTA-PEG2Nle-CycMSHhex and 64Cu-NOTA-AocNle-CycMSHhex were readily prepared with more than 90% radiolabeling yields and showed MC1R-specific binding on B16/F10 cells. 64Cu-NOTA-PEG2Nle-CycMSHhex exhibited higher tumor uptake than 64Cu-NOTA-AocNle-CycMSHhex at 0.5, 2, 4, and 24 h post-injection. The tumor uptake of 64Cu-NOTA-PEG2Nle-CycMSHhex was 16.23 ± 0.42, 19.59 ± 1.48, 12.83 ± 1.69, and 8.78 ± 2.29% ID/g at 0.5, 2, 4, and 24 h post-injection, respectively. Normal organ uptake of 64Cu-NOTA-PEG2Nle-CycMSHhex was lower than 2% ID/g at 2 h post-injection except for kidney uptake. The renal uptake of 64Cu-NOTA-PEG2Nle-CycMSHhex was 3.66 ± 0.52, 3.27 ± 0.52, and 1.47 ± 0.56 ID/g at 2, 4, and 24 h post-injection, respectively. 64Cu-NOTA-PEG2Nle-CycMSHhex showed high tumor to normal organ uptake ratios after 2 h post-injection. The B16/F10 melanoma lesions could be clearly visualized by positron emission tomography (PET) using 64Cu-NOTA-PEG2Nle-CycMSHhex as an imaging probe at 2 h post-injection. High tumor uptake and low kidney uptake of 64Cu-NOTA-PEG2Nle-CycMSHhex underscored its potential as an MC1R-targeted theranostic peptide for melanoma imaging and therapy.

The Effect of Albumin-Binding Moiety on Tumor Targeting and Biodistribution Properties of 67Ga-Labeled Albumin Binder-Conjugated Alpha-Melanocyte-Stimulating Hormone Peptides.

Background: The purpose of this study was to examine the effect of 4-p-(tolyl)butyric acid as an albumin-binding (ALB) moiety on tumor targeting and biodistribution properties of 67Ga-labeled albumin binder-conjugated alpha-melanocyte-stimulating hormone peptides. Materials and Methods: DOTA-Lys(ALB)-G/GG/GGG-Nle-CycMSHhex {1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-Lys(ALB)-Gly/GlyGly/GlyGlyGly-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} were synthesized with 4-p-(tolyl)butyric acid serving as an ALB moiety. The melanocortin-1 receptor (MC1R)-binding affinities of the peptides were determined on B16/F10 melanoma cells. The biodistribution of 67Ga-DOTA-Lys(ALB)-G/GG/GGG-Nle-CycMSHhex was examined on B16/F10 melanoma-bearing C57 mice at 2 h postinjection to select a lead peptide for further evaluation. The melanoma targeting and imaging properties of 67Ga-DOTA-Lys(ALB)-GGNle-CycMSHhex {67Ga-ALB-G2} were determined on B16/F10 melanoma-bearing C57 mice. Results: The IC50 value of DOTA-Lys(ALB)-G/GG/GGG-Nle-CycMSHhex {ALB-G1, ALB-G2, ALB-G3} was 0.67 ± 0.07, 0.5 ± 0.09 and 0.51 ± 0.03 nM on B16/F10 cells, respectively. 67Ga-ALB-G2 was further evaluated as a lead peptide because of its higher tumor uptake (30.25 ± 3.24%ID/g) and lower kidney uptake (7.09 ± 2.22%ID/g) than 67Ga-ALB-G1 and 67Ga-ALB-G3 at 2 h postinjection. The B16/F10 melanoma uptake of 67Ga-ALB-G2 was 15.64 ± 4.55, 30.25 ± 3.24, 26.76 ± 3.23, and 10.71 ± 1.21%ID/g at 0.5, 2, 4, and 24 h postinjection, respectively. The B16/F10 melanoma lesions were clearly visualized by SPECT/CT using 67Ga-ALB-G2 as an imaging probe at 2 h postinjection. Conclusions: The introduction of 4-p-(tolyl)butyric acid as an ALB moiety increased the blood retention, and resulted in higher tumor/kidney ratio of 67Ga-ALB-G2 as compared with its counterpart without an albumin binder. However, the resulting high uptake of 67Ga-ALB-G2 in blood and liver need to be further reduced to facilitate its therapeutic application when replacing 67Ga with therapeutic radionuclides.

A Two-Step Phenotypic Parameter Measurement Strategy for Overlapped Grapes under Different Light Conditions.

Phenotypic characteristics of fruit particles, such as projection area, can reflect the growth status and physiological changes of grapes. However, complex backgrounds and overlaps always constrain accurate grape border recognition and detection of fruit particles. Therefore, this paper proposes a two-step phenotypic parameter measurement to calculate areas of overlapped grape particles. These two steps contain particle edge detection and contour fitting. For particle edge detection, an improved HED network is introduced. It makes full use of outputs of each convolutional layer, introduces Dice coefficients to original weighted cross-entropy loss function, and applies image pyramids to achieve multi-scale image edge detection. For contour fitting, an iterative least squares ellipse fitting and region growth algorithm is proposed to calculate the area of grapes. Experiments showed that in the edge detection step, compared with current prevalent methods including Canny, HED, and DeepEdge, the improved HED was able to extract the edges of detected fruit particles more clearly, accurately, and efficiently. It could also detect overlapping grape contours more completely. In the shape-fitting step, our method achieved an average error of 1.5% in grape area estimation. Therefore, this study provides convenient means and measures for extraction of grape phenotype characteristics and the grape growth law.

Advances in Receptor-Targeted Radiolabeled Peptides for Melanoma Imaging and Therapy.

Melanocortin-1 receptor (MC1R) and very late antigen-4 (VLA-4, integrin α4ß1) are 2 attractive molecular targets for developing peptide radiopharmaceuticals for melanoma imaging and therapy. MC1R- and VLA-4-targeting peptides and peptide-conjugated Cornell prime dots (C' dots) can serve as delivery vehicles to target both diagnostic and therapeutic radionuclides to melanoma cells for imaging and therapy. This review highlights the advances of MC1R- and VLA-4-targeted radiolabeled peptides and peptide-conjugated C' dots for melanoma imaging and therapy. The promising preclinical and clinical results of these new peptide radiopharmaceuticals present an optimistic outlook for clinical translation into receptor-targeting melanoma imaging and radionuclide therapy in the future.

Facile preparation of a novel Ga-67-labeled NODAGA-conjugated lactam-cyclized alpha-MSH peptide at room temperature for melanoma targeting.

In this study, the melanoma targeting property of 67Ga-NODAGA-GGNle-CycMSHhex {1,4,7-triazacyclononane,1-gluteric acid-4,7-acetic acid-GlyGlyNle-c[Asp-His-D-Phe-Arg-Trp-Lys]-CONH2} was determined on B16/F10 melanoma-bearing C57 mice to demonstrate the feasibility of NODAGA as a radiometal chelator for facile room temperature radiolabeling of NODAGA-GGNle-CycMSHhex. The IC50 value of NODAGA-GGNle-CycMSHhex was 0.87 ± 0.12 nM on B16/F10 melanoma cells. 67Ga-NODAGA-GGNle-CycMSHhex was readily prepared at room temperature with greater than 98% radiolabeling yield and displayed MC1R-specific binding on B16/F10 melanoma cells. The B16/F10 melanoma uptake of 67Ga-NODAGA-GGNle-CycMSHhex was 10.31 ± 0.78, 14.96 ± 1.34, 13.7 ± 3.33 and 10.4 ± 2.2% ID/g at 0.5, 2, 4 and 24 h post-injection, respectively. Approximately 85% of the injected dose was cleared out the body via urinary system at 2 h post-injection. 67Ga-NODAGA-GGNle-CycMSHhex showed high tumor/blood, tumor/muscle and tumor/skin uptake ratios after 2 h post-injection. Overall, 67Ga-NODAGA-GGNle-CycMSHhex could be easily prepared at room temperature and exhibited favorable melanoma targeting property, suggesting the potential use of NODAGA as a radiometal chelator for facile room temperature radiolabeling of α-MSH peptides.

Novel [99mTc]-Tricarbonyl-NOTA-Conjugated Lactam-Cyclized Alpha-MSH Peptide with Enhanced Melanoma Uptake and Reduced Renal Uptake.

The purpose of this study was to examine the melanoma targeting and imaging properties of 99mTc(CO)3-NOTA-GGNle-CycMSHhex {1,4,7-triazacyclononane-1,4,7-triyl-triacetic acid-GlyGlyNle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} and 99mTc(CO)3-NODAGA-GGNle-CycMSHhex {1,4,7-triazacyclononane,1-gluteric acid-4,7-acetic acid-GlyGlyNle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} on B16/F10 melanoma-bearing C57 mice to demonstrate the feasibility of NOTA/NODAGA as metal chelators for 99mTc(CO)3+ radiolabeling. NOTA/NODAGA-GGNle-CycMSHhex were synthesized using fluorenylmethoxycarbonyl (Fmoc) chemistry. The melanocortin-1 (MC1) receptor binding affinities of the peptides were determined on B16/F10 melanoma cells. The biodistribution of 99mTc(CO)3-NOTA-GGNle-CycMSHhex and 99mTc(CO)3-NODAGA-GGNle-CycMSHhex were determined on B16/F10 melanoma-bearing C57 mice at 2 h postinjection to select a lead peptide for further evaluation. The melanoma targeting and imaging properties of 99mTc(CO)3-NOTA-GGNle-CycMSHhex and 99mTc(CO)3-NODAGA-GGNle-CycMSHhex were determined on B16/F10 melanoma-bearing C57 mice. The IC50 values of NOTA/NODAGA-GGNle-CycMSHhex were 0.8 ± 0.1 and 0.9 ± 0.1 nM on B16/F10 cells. 99mTc(CO)3-NOTA-GGNle-CycMSHhex and 99mTc(CO)3-NODAGA-GGNle-CycMSHhex were readily prepared via the [99mTc(CO)3(OH2)3]+ intermediate and displayed MC1R-specific binding on B16/F10 cells. 99mTc(CO)3-NOTA-GGNle-CycMSHhex was further evaluated as a lead peptide because of its higher tumor uptake (19.76 ± 3.62% ID/g) and lower kidney uptake (1.59 ± 0.52% ID/g) at 2 h postinjection than 99mTc(CO)3-NODAGA-GGNle-CycMSHhex. The B16/F10 melanoma uptake of 99mTc(CO)3-NOTA-GGNle-CycMSHhex was 16.07 ± 4.47, 19.76 ± 3.62, 11.30 ± 2.81, and 3.16 ± 2.28% ID/g at 0.5, 2, 4, and 24 h postinjection, respectively. 99mTc(CO)3-NOTA-GGNle-CycMSHhex showed high tumor to normal organ uptake ratios after 2 h postinjection. The B16/F10 melanoma lesions were clearly visualized by SPECT/CT using 99mTc(CO)3-NOTA-GGNle-CycMSHhex as an imaging probe at 2 h postinjection. High tumor uptake, low kidney uptake, and fast urinary clearance of 99mTc(CO)3-NOTA-GGNle-CycMSHhex highlighted its potential for melanoma imaging and facilitated the evaluation of 188Re(CO)3-NOTA-GGNle-CycMSHhex for melanoma therapy.

Melanoma-Targeting Property of Y-90-Labeled Lactam-Cyclized α-Melanocyte-Stimulating Hormone Peptide.

Purpose: The purpose of this study was to evaluate melanoma-targeting property of 90Y-DOTA-GGNle-CycMSHhex to facilitate its potential therapeutic application. Materials and Methods: DOTA-GGNle-CycMSHhex was synthesized and readily labeled with 90Y in 0.25 M NH4Ac-buffered solution to generate 90Y-DOTA-GGNle-CycMSHhex. The specific receptor binding, internalization, and efflux of 90Y-DOTA-GGNle-CycMSHhex were determined on B16/F10 murine melanoma cells. The biodistribution property of 90Y-DOTA-GGNle-CycMSHhex was examined on B16/F10 melanoma-bearing C57 mice. Results: 90Y-DOTA-GGNle-CycMSHhex displayed receptor-specific binding, rapid internalization, and prolonged efflux on B16/F10 melanoma cells. 90Y-DOTA-GGNle-CycMSHhex exhibited high uptake and prolonged retention in melanoma, and fast urinary clearance on B16/F10 melanoma-bearing C57 mice. The B16/F10 tumor uptake was 20.73% ± 7.99%, 19.93% ± 5.73%, 14.8% ± 4.61%, and 6.69% ± 1.85% ID/g at 0.5, 2, 4, and 24 h postinjection, respectively. Conclusions: 90Y-DOTA-GGNle-CycMSHhex displayed melanocortin-1 receptor (MC1R) targeting and specificity on B16/F10 melanoma cells and tumors. The favorable melanoma-targeting property and fast urinary clearance of 90Y-DOTA-GGNle-CycMSHhex warranted its evaluation for melanoma therapy in future studies.

Evaluation of a Novel Pb-203-Labeled Lactam-Cyclized Alpha-Melanocyte-Stimulating Hormone Peptide for Melanoma Targeting.

The purpose of this study is to examine the melanocortin-1 receptor (MC1R) targeting and specificity of 203Pb-DOTA-GGNle-CycMSHhex in melanoma cells and tumors to facilitate its potential therapeutic application when labeled with 212Pb. The MC1R-specific targeting and imaging properties of 203Pb-DOTA-GGNle-CycMSHhex were determined on B16/F1 and B16/F10 murine melanoma cells and in B16/F1 flank melanoma-, B16/F10 flank melanoma-, and B16/F10 pulmonary metastatic melanoma-bearing C57 mice. 203Pb-DOTA-GGNle-CycMSHhex displayed MC1R-specific binding on B16/F1 and B16/F10 melanoma cells and tumors. B16/F1 flank melanoma, B16/F10 flank melanoma, and B16/F10 pulmonary metastatic melanoma lesions could be clearly imaged by single photon emission computed tomography (SPECT) using 203Pb-DOTA-GGNle-CycMSHhex as an imaging probe. The favorable melanoma targeting and imaging properties highlighted the potential of 203Pb-DOTA-GGNle-CycMSHhex as a MC1R-targeting melanoma imaging probe and warranted the evaluation of 212Pb-DOTA-GGNle-CycMSHhex for melanoma therapy in future studies.

68Ga-DOTA-GGNle-CycMSHhex targets the melanocortin-1 receptor for melanoma imaging.

Melanocortin-1 receptor (MC1R) is a molecular target for melanoma imaging and therapy because of its overexpression on rodent and human melanoma cells. Here, we evaluated the MC1R targeting and specificity of 68Ga-DOTA-GGNle-CycMSHhex and Cy5.5-GGNle-CycMSHhex using murine and human melanoma cells, and murine and xenografted tumors. 68Ga-DOTA-GGNle-CycMSHhex was used first in human as an imaging probe to evaluate the possibility of radionuclide therapy in patients with advanced-stage melanoma. 68Ga-DOTA-GGNle-CycMSHhex and Cy5.5-GGNle-CycMSHhex displayed MC1R-specific targeting properties in murine and human melanoma cells, as well as in murine melanoma and human melanoma-xenografted tumors. Both B16/F10 and M21 melanoma lesions could be easily imaged by positron emission tomography using 68Ga-DOTA-GGNle-CycMSHhex The first-in-human images of melanoma brain metastases in patients demonstrated the clinical relevance of MC1R as a molecular target for melanoma imaging, highlighting the potential of 68Ga-DOTA-GGNle-CycMSHhex as an MC1R-targeting melanoma imaging probe and underscoring the need to develop MC1R-targeting therapeutic agents for treating patients with metastatic melanoma.

Matched-pair, 86Y/90Y-labeled, bivalent RGD/bombesin antagonist, [RGD-Glu-[DO3A]-6-Ahx-RM2], as a potential theranostic agent for prostate cancer.

INTRODUCTION: In this study, we describe development of a true matched-pair theranostic agent that is able to target the αVß3 integrin and the gastrin releasing peptide receptor (GRPR). We herein describe methods to metallate and characterize the new conjugate and to validate its biological efficacy by in vitro and in vivo methods. METHODS: We have previously described the development of [RGD-Glu-6Ahx-RM2] (where RGD: Arg-Gly-Asp; Glu: glutamic acid; 6-Ahx: 6-amino hexanoic acid; RM2: (D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2)) that has been conjugated to a DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) bifunctional chelating agent (BFCA) to afford [RGD-Glu-[DO3A]-6-Ahx-RM2] peptide. In this study, we have radiolabeled [RGD-Glu-[DO3A]-6-Ahx-RM2] peptide with 86Y or 90Y. Natural-metallated (natY) conjugates were assessed for binding affinity for the αVß3 integrin or GRPR in human glioblastoma U87-MG and prostate PC-3 cell lines, respectively. The effective stability of the new tracers was also evaluated prior to in vivo evaluation in normal CF-1 mice and SCID mice bearing xenografted tumors. RESULTS: Competitive displacement binding assays in PC-3 cells showed high binding affinity for the GRPR (IC50, 5.65 ±â€¯0.00 nM). On the other hand, competitive displacement binding assays in U87-MG cells revealed only moderate binding to the αVß3 integrin (IC50, 346 ±â€¯5.30 nM). Biodistribution studies in PC-3 tumor-bearing mice [RGD-Glu-[[90Y]Y-DO3A]-6-Ahx-RM2] showed high tumor uptake (8.70 ±â€¯0.35%ID/g at 1 h post-intravenous injection) and retention of tracer (5.28 ±â€¯0.12%ID/g) at 24 h post-intravenous injection. Micro-positron emission tomography (microPET) in PC-3 tumor-bearing mice using [RGD-Glu-[[86Y]Y-DO3A]-6-Ahx-RM2] correlated well with biodistribution investigations over the various time points that were studied. CONCLUSIONS: The [RGD-Glu-[[86Y]Y-DO3A]-6-Ahx-RM2] and [RGD-Glu-[[90Y]Y-DO3A]-6-Ahx-RM2] matched-pair conjugates described herein exhibit favorable microPET and pharmacokinetic profiles and merit further investigations for molecular imaging and/or therapeutic evaluation in larger animal models and potentially humans. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: The theranostic, heterobivalent, agents described herein perform comparably with other mono- and multivalent conjugates we have reported and offer the potential of improved sensitivity for detecting prostate cancer cells that might exhibit differing profiles of receptor expression on tumor cells in human patients.

Role of an isoform-specific residue at the calmodulin-heme (NO synthase) interface in the FMN - heme electron transfer.

The interface between calmodulin (CaM) and the NO synthase (NOS) heme domain is the least characterized interprotein interface that the NOS isoforms must traverse through during catalysis. Our previous molecular dynamics simulations predicted a salt bridge between K497 in human inducible NOS (iNOS) heme domain and D118(CaM). Herein, the FMN - heme interdomain electron transfer (IET) rate was found to be notably decreased by charge-reversal mutation, while the IET in the iNOS K497D mutant is significantly restored by the CaM D118K mutation. The results of wild-type protein with added synthetic peptides further demonstrate the critical nature of K497 relative to the rest of the peptide sequence in modulating the IET. These data provide definitive evidence supporting the regulatory role of the isoform-specific K497 residue.

A docked state conformational dynamics model to explain the ionic strength dependence of FMN - heme electron transfer in nitric oxide synthase.

The FMN-heme interdomain electron transfer (IET) in nitric oxide synthase (NOS) is a key stage of the electron transport chain, which supplies the catalytic heme site(s) with the NADPH-derived electrons. While there is a recognition that this IET depends on both the electron tunneling and the conformational dynamics, the detailed mechanism remains unclear. In this work, the IET kinetics were measured by laser flash photolysis for a bidomain oxygenase/FMN (oxyFMN) construct of human inducible NOS (iNOS) over the ionic strength range from 0.1 to 0.5 M. The forward (heme → FMN, kETf) and backward (FMN → heme, kETb) intrinsic IET rate constants were determined from the analysis of the observed IET rates using the additional information regarding the conformational dynamics obtained from the FMN fluorescence lifetime measurements and theoretical estimates. Both kETf and kETb exhibit a bell-shaped dependence on the ionic strength, I, with the maximum rates corresponding to I ~ 0.2 M. This dependence was explained using a new model, which considers the effect of formation of pairs between the protein surface charged residues and solution ions on the docked state dynamics. The trial simulations of the intrinsic IET rate dependences using this model show that the data can be reproduced using reasonable energetic, structural, and chemical parameters. The suggested model can explain both the monophasic and biphasic ionic strength dependences and can be used to rationalize the interprotein/interdomain electron transfer rates for other types of protein systems where the docked state is sufficiently long-lived.

GABAA Receptor Availability Changes Underlie Symptoms in Isolated Cervical Dystonia.

GABAA receptor availability changes within sensorimotor regions have been reported in some isolated forms of dystonia. Whether similar abnormalities underlie symptoms in cervical dystonia is not known. In the present study, a total of 15 cervical dystonia patients and 15 age- and sex-matched controls underwent 11C-flumazenil PET/CT scanning. The density of available GABAA receptors was estimated using a Simplified Reference Tissue Model 2. Group differences were evaluated using a two-sample T-test, and correlations with dystonia severity, as measured by the Toronto Western Spasmodic Torticollis Rating Scale, and disease duration were evaluated using a regression analysis. Voxel-based analyses revealed increased GABAA availability within the right precentral gyrus in brain motor regions previously associated with head turning and the left parahippocampal gyrus. GABAA availability within the bilateral cerebellum was negatively correlated with dystonia severity, and GABAA availability within the right thalamus and a variety of cerebellar and cortical regions were negatively correlated with disease duration. While GABAA availability changes within primary motor areas could represent a partial compensatory response to loss of inhibition within sensorimotor network, GABAergic signaling impairment within the cerebellum may be a key contributor to dystonia severity.