Timothy J. Hoffman

[64Cu-NOTA-8-Aoc-BBN(7-14)NH2] targeting vector for positron-emission tomography imaging of gastrin-releasing peptide receptor-expressing tissues

Radiolabeled peptides hold promise as diagnostic/therapeutic targeting vectors for specific human cancers. We report the design and development of a targeting vector, [64Cu-NOTA-8-Aoc-BBN(7-14)NH2] (NOTA = 1,4,7-triazacyclononane-1,4,7-triacetic acid, 8-Aoc = 8-aminooctanoic acid, and BBN = bombesin), having very high selectivity and affinity for the gastrin-releasing peptide receptor (GRPr). GRPrs are expressed on a variety of human cancers, including breast, lung, pancreatic, and prostate, making this a viable approach toward site-directed localization or therapy of these human diseases. In this study, [NOTA-X-BBN(7-14)NH2] conjugates were synthesized, where X = a specific pharmacokinetic modifier. The IC50 of [NOTA-8-Aoc-BBN(7-14)NH2] was determined by a competitive displacement cell-binding assay in PC-3 human prostate cancer cells using 125I-[Tyr4]-BBN as the displacement ligand. An IC50 of 3.1 ± 0.5 nM was obtained, demonstrating high binding affinity of [NOTA-8-Aoc-BBN] for the GRPr. [64Cu-NOTA-X-BBN] conjugates were prepared by the reaction of 64CuCl2 with peptides in buffered aqueous solution. In vivo studies of [64Cu-NOTA-8-Aoc-BBN(7-14)NH2] in tumor-bearing PC-3 mouse models indicated very high affinity of conjugate for the GRPr. Uptake of conjugate in tumor was 3.58 ± 0.70% injected dose (ID) per g at 1 h postintravenous injection (p.i.). Minimal accumulation of radioactivity in liver tissue (1.58 ± 0.40% ID per g, 1 h p.i.) is indicative of rapid renal-urinary excretion and suggests very high in vivo kinetic stability of [64Cu-NOTA-8-Aoc-BBN(7-14)NH2] with little or no in vivo dissociation of 64Cu2+ from the NOTA chelator. Kidney accumulation at 1 h p.i. was 3.79 ± 1.09% ID per g. Molecular imaging studies in GRPr-expressing tumor models produced high-contrast, high-quality micro-positron-emission tomography images.

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, β+: 18%, Eβ+max = 653 keV; β−: 37%, Eβ−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.

Gastrin releasing peptide (GRP) receptor targeted radiopharmaceuticals: a concise update.

The gastrin releasing peptide (GRP) receptor is becoming an increasingly attractive target for development of new radiolabeled peptides with diagnostic and therapeutic potential. The attractiveness of the GRP receptor as a target is based upon the functional expression of GRP receptors in several tumors of neuroendocrine origin including prostate, breast, and small cell lung cancer. This concise review outlines some of the efforts currently underway to develop new GRP receptor specific radiopharmaceuticals by employing a variety of radiometal chelation systems.

99mTc-propylene amine oxime (99mTc-PnAO); a potential brain radiopharmaceutical

Propylene amine oxime (PnAO) forms a neutral lipid soluble complex with 99mTc. 99mTc-PnAO can be prepared by simple reduction of 99mTc-pertechnetate with stannous ion in the presence of excess PnAO in saline at or near neutral pH. This agent will passively penetrate biological membranes including the intact blood-brain barrier (BBB) as evidenced by the high brain uptake observed shortly after IV injection. The first-pass extraction efficiency in baboons was estimated to be 80% at normal blood flow. This agent or a derivatized form of 99mTc-PnAO may be useful in assessment of regional cerebral blood flow (rCBF) in humans.

Melanoma Therapy via Peptide-Targeted α-Radiation

Purpose: The therapeutic efficacy of a unique melanoma-targeting peptide conjugated with an in vivo generated α-particle-emitting radionuclide was evaluated in the B16/F1 mouse melanoma animal model. α-Radiation is densely ionizing, resulting in high concentrations of destructive radicals and irreparable DNA double-strand breaks. This high linear energy transfer overcomes radiation-resistant tumor cells and oxygen effects resulting in potentially high therapeutic indices in tumors such as melanoma. Experimental Design: The melanoma targeting peptide, 1,4,7,10-tetraazacyclodecane-1,4,7,10-tetraacetic acid (DOTA)-Re(Arg11)CCMSH, was radiolabeled with 212Pb, the parent of 212Bi, which decays via α and β decay. Biodistribution and therapy studies were done in the B16/F1 melanoma-bearing C57 mouse flank tumor model. Results:212Pb[DOTA]-Re(Arg11)CCMSH exhibited rapid tumor uptake and extended retention coupled with rapid whole body disappearance. Radiation dose delivered to the tumor was estimated to be 61 cGy/μCi 212Pb administered. Treatment of melanoma-bearing mice with 50, 100, and 200 μCi of 212Pb[DOTA]-Re(Arg11)CCMSH extended their mean survival to 22, 28, and 49.8 days, respectively, compared with the 14.6-day mean survival of the placebo control group. Forty-five percent of the mice receiving 200 μCi doses survived the study disease-free. Conclusions: Treatment of B16/F1 murine melanoma–bearing mice with 212Pb[DOTA]-Re(Arg11)CCMSH significantly decreased tumor growth rates resulting in extended mean survival times, and in many cases, complete remission of disease. 212Pb-DOTA-Re(Arg11)CCMSH seems to be a very promising radiopharmaceutical for targeted radionuclide therapy of melanoma.

In vivo evaluation of 188Re-labeled alpha-melanocyte stimulating hormone peptide analogs for melanoma therapy

The purpose of our study was to optimize melanoma tumor uptake of 188Re‐CCMSH and reduce its nonspecific kidney retention. Nephrotoxicity is often a serious problem associated with targeted radiotherapy, therefore, increasing the tumor/kidney uptake ratio of 188Re‐CCMSH is crucial for optimizing its therapeutic efficacy. Structural modification of the peptide and amino acid co‐infusion were investigated as strategies to improve the tumor/kidney uptake ratio of 188Re‐CCMSH. The substitution of Lys 11 with Arg 11 was examined to determine if removal of lysine from the peptide would improve kidney clearance without sacrificing tumor uptake. The pharmacokinetics of 188Re‐CCMSH and 188Re‐(Arg11)CCMSH were determined in B16/F1 murine melanoma‐bearing C57 mice. Tumor uptake values of 188Re‐CCMSH and 188Re‐(Arg11)CCMSH were 15.03 ± 5.20% ID/g and 20.44 ± 1.91% ID/g at 1 hr postinjection and 1.94 ± 0.47% ID/g and 3.50 ± 2.32% ID/g at 24 hr postinjection. Renal retention of 188Re‐(Arg11)CCMSH was 11.79 ± 1.29 ID/g and 3.67 ± 0.51 ID/g at 1 hr and 4 hr postinjection, which was a greater than 50% reduction compared to 188Re‐CCMSH. The Arg for Lys substitution in 188Re‐(Arg11)CCMSH resulted in improved tumor uptake and reduced kidney retention. Renal retention of both 188Re‐CCMSH and 188Re‐(Arg11)CCMSH were significantly reduced by co‐injection of 20 mg of L‐lysine, L‐arginine and a combination of L‐lysine:L‐arginine. Tumor/kidney uptake values for 188Re‐CCMSH and 188Re‐(Arg11)CCMSH were maximally reduced by 52.9% and 46.3%, respectively. However, even with amino acid co‐injection, the tumor/kidney ratio of 188Re‐CCMSH was lower than that of 188Re‐(Arg11)CCMSH. Improved tumor uptake and reduced kidney retention of 188Re‐(Arg11)CCMSH will facilitate targeted irradiation of melanoma tumors while minimizing the dose to the kidneys.

Development of an in vitro model for assessing the in vivo stability of lanthanide chelates

An in vitro model was developed to evaluate the in vivo stability of lanthanide polyaminocarboxylate complexes. The ligand-to-metal ratios for the chelates EDTA, CDTA, DTPA, MA-DTPA (monoamide-DTPA) and DOTA with the lanthanides lanthanum, samarium, and lutetium were optimized to achieve > or = 98% complexation yield for the resultant radiolanthanide complexes. The exchange of the radiolanthanides from their EDTA, CDTA, DTPA, MA-DTPA and DOTA complexes with Ca(2+) was determined by in vitro adsorption and in vitro column studies using hydroxyapatite (HA), an in vitro bone model. In vitro serum stability of these radiolanthanide complexes was used as an additional indicator of in vivo stability, although the mechanism of instability in serum will be different than with bone. The in vitro studies were consistent with the expected findings that the smallest lanthanide (Lu) formed the most stable complexes. In vivo studies were done to validate the in vitro model. Biodistribution studies in normal CF-1 mice showed that in vivo stability of the complex (i.e., the more lanthanide remaining in complex form) could be assessed by a combination of the urinary, bone and liver uptake. For example, biodistribution studies demonstrate that high urinary excretion correlated with complex stability, while high liver plus bone uptake correlated with complex instability. The urinary excretion of the EDTA complexes decreased from (177)Lu to (140)La indicating a loss in stability in the direction of (140)La, consistent with the in vitro studies. The more stable a lanthanide complex is, the lower its exchange with HA in vitro will be, and the lower its combined bone plus liver uptake and higher its urinary excretion will be in vivo. This investigation indicates that the in vivo stability can be determined by a screening method that measures the degree of exchange from the lanthanide chelate with hydroxyapatite (HA) and its serum stability.

Optimization, biological evaluation and microPET imaging of copper-64-labeled bombesin agonists, [64Cu-NO2A-(X)-BBN(7–14)NH2], in a prostate tumor xenografted mouse model☆

UNLABELLED Gastrin-releasing peptide receptors (GRPr) are a member of the bombesin (BBN) receptor family. GRPr are expressed in high numbers on specific human cancers, including human prostate cancer. Therefore, copper-64 ((64)Cu) radiolabeled BBN(7-14)NH(2) conjugates could have potential for diagnosis of human prostate cancer via positron-emission tomography (PET). The aim of this study was to produce [(64)Cu-NO2A-(X)-BBN(7-14)NH(2)] conjugates for prostate cancer imaging, where X=pharmacokinetic modifier (beta-alanine, 5-aminovaleric acid, 6-aminohexanoic acid, 8-aminooctanoic acid, 9-aminonanoic acid or para-aminobenzoic acid) and NO2A=1,4,7-triazacyclononane-1,4-diacetic acid [a derivative of NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid)]. METHODS [(X)-BBN(7-14)NH(2)] Conjugates were synthesized by solid-phase peptide synthesis (SPPS), after which NOTA was added via manual conjugation. The new peptide conjugates were radiolabeled with (64)Cu radionuclide. The receptor-binding affinity was determined in human prostate PC-3 cells, and tumor-targeting efficacy was determined in PC-3 tumor-bearing severely combined immunodeficient (SCID) mice. Whole-body maximum intensity microPET/CT images of PC-3 tumor-bearing SCID mice were obtained 18 h postinjection (pi). RESULTS Competitive binding assays in PC-3 cells indicated high receptor-binding affinity for the [NO2A-(X)-BBN(7-14)NH(2)] and [(nat)Cu-NO2A-(X)-BBN(7-14)NH(2)] conjugates. In vivo biodistribution studies of the [(64)Cu-NO2A-(X)-BBN(7-14)NH(2)] conjugates at 1, 4 and 24 h pi showed very high uptake of the tracer in GRPr-positive tissue with little accumulation and retention in nontarget tissues. High-quality, high-contrast microPET images were obtained, with xenografted tumors being clearly visible at 18 h pi. CONCLUSIONS NO2A chelator sufficiently stabilizes copper(II) radiometal under in vivo conditions, producing conjugates with very high uptake and retention in targeted GRPr. Preclinical evaluation of these new peptide conjugates in tumor-bearing mice provides some impetus for clinical evaluation in human patients.

Radiometallated receptor-avid peptide conjugates for specific in vivo targeting of cancer cells

New receptor-avid radiotracers are being developed for site-specific in vivo targeting of a myriad of receptors expressed on cancer cells. This review exemplifies strategies being used to design radiometallated peptide conjugates that maximize uptake in tumors and optimize their in vivo pharmacokinetic properties. Efforts to produce synthetic peptide analogues that target the following three receptor systems are highlighted: Gastrin releasing peptide (GRP), alpha-melanocyte stimulating hormone (alpha-MSH), and guanylate cyclase-C (GC-C) receptors.

In vitro and in vivo analysis of [64Cu-NO2A-8-Aoc-BBN(7–14)NH2]: a site-directed radiopharmaceutical for positron-emission tomography imaging of T-47D human breast cancer tumors

INTRODUCTION Human breast cancer, from which the T-47D cell line was derived, is known to overexpress the gastrin-releasing peptide receptor (GRPR) in some cases. Bombesin (BBN), an agonist for the GRPR, has been appended with a radionuclide capable of positron-emission tomography (PET) imaging and therapy. (64)Cu-NO2A-8-Aoc-BBN(7-14)NH(2) (NO2A=1,4,7-triazacyclononane-1,4-diacetate) has produced high-quality microPET images of GRPR-positive breast cancer xenografted tumors in mice. METHODS The imaging probe was synthesized by solid-phase peptide synthesis followed by manual conjugation of the 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) bifunctional chelator and radiolabeling in aqueous solution. The radiolabeled conjugate was subjected to in vitro and in vivo studies to determine its specificity for the GRPR and its pharmacokinetic profile. A T-47D tumor-bearing mouse was imaged with microPET/CT and microMRI imaging. RESULTS The (64)Cu-NO2A-8-Aoc-BBN(7-14)NH(2) targeting vector was determined to specifically localize in GRPR-positive tissue. Accumulation was observed in the tumor in sufficient quantities to allow for identification of tumors in microPET imaging procedures. For example, uptake and retention in T-47D xenografts at 1, 4 and 24 h were determined to be 2.27+/-0.08, 1.35+/-0.14 and 0.28+/-0.07 % ID/g, respectively. CONCLUSIONS The (64)Cu-NO2A-8-Aoc-BBN(7-14)NH(2) produced high-quality microPET images. The pharmacokinetic profile justifies investigation of this bioconjugate as a potentially useful diagnostic/therapeutic agent. Additionally, the bioconjugate would serve as a good starting point for modification and optimization of similar agents to maximize tumor uptake and minimize nontarget accumulation.