Synthesis and characterization of Bombesin derivatives with potential applications as nuclear medicine imaging/therapeutic agents [abstract]

Abstract only availableFaculty Mentor: Charles J. Smith, RadiologyThe use of tissue specific radiopharmaceuticals presents great promise for diagnostic and therapeutic applications in a number of human cancers. Gastrin-releasing peptide (GRP) receptors are known to be over-expressed on a variety of malignancies including breast, gastric, colon, pancreatic, prostate, and small-cell lung cancers. Experimental work with bombesin (BBN), an amphibian analogue to mammalian GRP, has demonstrated the ability of BBN to bind, with high affinity and specificity, to the GRP receptor. The use of a bifunctional chelating agent (BFCA) allows biologically active molecules to maintain receptor affinity while at the same time complexing a radionuclide. Spacer groups separating the BFCA and biological vector allow for fine tuning of the pharmacokinetics. Bombesin conjugates effectively complexing radioactive copper (i.e. Cu-64; t1/2=12.7h, Eγ=1345.8keV, Eβ-=578keV, Eβ+=651keV) posses potential as imaging agents for diagnostic positron emission tomography (PET) and therapeutic applications.Recently our research group has focused efforts on developing new BBN derivatives of Triaza (1,4,7-triazacyclononane) or functionalized derivatives of Triaza for complexing of specific radionuclides. Our laboratory has previously reported on a series of new Triaza-BBN conjugates. A derivative of this Triaza ligand system, 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), has been synthesized by alkylation of the secondary amines of triaza by α-chloroacetic acid prior to conjugation to the biologically active BBN targeting vector. Bombesin conjugates were derived of the form, NOTA-X-BBN (X = ßAla, GGG, SSS). Synthesis of the unligated BBN [7-14] peptide with spacer group was conducted by Fmoc-protected solid-phase peptide synthesis (SPPS). The bombesin constructs were purified prior to conjugation of the ligand framework by means of reverse phase-high performance liquid chromatography (RP-HPLC). The NOTA ligand was conjugated to the N-terminus of the peptide by means of an activated ester derived from N-hydroxysulfosuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl in aqueous solution. The final NOTA-X-BBN derivatives were purified by RP-HPLC and confirmed via electrospray ionization-mass spectrometry (ESI-MS). Further studies to evaluate the ability of the derivatives to complex Cu-64 are underway. Subsequent studies will be conducted to evaluate the in vitro and in vivo characteristics of the radiopharmaceutical

Evaluation of a new series of copper-64-nota-bombesin targeted radiopharmecuticals with PET imaging potential [abstract]

Abstract only availableTissue-specific radiopharmaceuticals having promise as diagnostic and therapeutic targeting vectors for human cancers is of significant interest. In recent years, our group and many others have focused upon design and development of targeting vectors having high selectivity and affinity for the gastrin-releasing peptide receptor (GRPr). GRP receptors are known to be over-expressed on a variety of malignancies including breast, lung, pancreatic, and prostate cancers. Bombesin (BBN), an amphibian analogue of mammalian GRP, has demonstrated the ability to bind with high affinity and specificity to the GRP receptor. In this study, we report the synthesis of the bifunctional chelating agent (BFCA) 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and conjugation to the biologically active BBN targeting vector. Bombesin conjugates were derived of the form, NOTA-X-BBN (X = GGG, SSS, ßAla, 5Ava, and 8Aoc). The unligated BBN [7-14] peptides with spacer groups were synthesized by solid-phase peptide synthesis (SPPS) and purified by reverse phase-high performance liquid chromatography (RP-HPLC). NOTA chelator was conjugated to the N-terminal primary amine of BBN manually. The final NOTA-X-BBN derivatives were purified by RP-HPLC and their chemical constitution verified. [64Cu-NOTA-X-BBN]-conjugates were prepared by addition of 64CuCl2 to a buffered solution containing the conjugate followed by purification via RP-HPLC. In vivo studies of [64Cu-NOTA-8-Aoc-BBN]-conjugates in normal CF-1 mice showed receptor-specific uptake in normal pancreatic tissue, an organ known to express the GRPr in very high numbers. Furthermore, studies in human-prostate tumor-bearing mice have demonstrated the ability of [64Cu-NOTA-8-Aoc-BBN] to undergo receptor-specific tumor uptake. Diagnostic positron emission tomography (PET) imaging using this conjugate was successful in resolving xenografted tumors in a mouse model. Both normal and tumor-bearing mice exhibited low liver accumulation of Cu-64, a known complication of 64Cu-containing radiopharmaceuticals. Further studies are needed to verify the efficacy of [64Cu-NOTA-8-Aoc-BBN] to target other human malignancies.Life Sciences Undergraduate Research Opportunity Progra

In vitro/in vivo assessment of novel 99mTc-bombesin conjugates in human cancer tissue [abstract]

Abstract only availableReceptor-specific, radiolabeled peptides are becoming increasingly popular as targeting vectors for the development of new diagnostic radiopharmaceuticals. The over-expression of certain receptors such as the gastrin releasing peptide receptor (GRPr) on human cancer cells makes this method of drug development a viable tool for tumor targeting in vivo. Breast, pancreatic, prostate, gastric, colon, and small-cell lung cancer have demonstrated GRPr expression. In this project, we have conjugated a diaminoproionic acid (DPR) bifunctional chelator to bombesin (BBN) peptide targeting vector by solid phase peptide synthesis. BBN is an analogue of human gastrin releasing peptide (GRP) that binds to the GRPr with high affinity and specificity. Conjugates of the general structure [DPR-(X)-BBN(7-14)NH2] (X = a series of amino acid pharmacokinetic modifiers) were purified by reverse-phase high-performance liquid chromatography and characterized by electrospray-ionization mass spectrometry. Radiolabeling investigations of with fac-[99mTc(CO)3(H2O)3]+ (Isolink®) provided for metallated conjugates of the following general structure: [99mTc(CO)3-DPR-(X)-BBN(7-14)NH2]. These new conjugates demonstrated the ability to target specific human tumors in rodent models. Subsequent radiolabeling studies of [DPR-(X)-BBN(7-14)NH2] with fac-[188Re(CO)3(H2O)3]+, the therapeutic surrogate precursor of Tc-99m, have given us the potential to treat specific human tumors via these new targeting vectors. Detailed radiolabeling protocols, in vitro cell binding studies, and in vivo biodistribution assays will be reported.Harry S. Truman Memorial VA Hospita

Anti-schistosomal immunity to core xylose/fucose in N-glycans

Schistosomiasis is a globally prevalent, debilitating disease that is poorly controlled by chemotherapy and for which no vaccine exists. While partial resistance in people may develop over time with repeated infections and treatments, some animals, including the brown rat (Rattus norvegicus), are only semi-permissive and have natural protection. To understand the basis of this protection, we explored the nature of the immune response in the brown rat to infection by Schistosoma mansoni. Infection leads to production of IgG to Infection leads to production of IgG to parasite glycoproteins parasite glycoproteins with complex-type N-glycans that contain a non-mammalian-type modification by core α2-Xylose and core α3-Fucose (core Xyl/Fuc). These epitopes are expressed on the surfaces of schistosomula and adult worms. Importantly, IgG to these epitopes can kill schistosomula by a complement-dependent process in vitro. Additionally, sera from both infected rhesus monkey and infected brown rat were capable of killing schistosomula in a manner inhibited by glycopeptides containing core Xyl/Fuc. These results demonstrate that protective antibodies to schistosome infections in brown rats and rhesus monkeys include IgG responses to the core Xyl/Fuc epitopes in surface-expressed N-glycans, and raise the potential of novel glyco-based vaccines that might be developed to combat this disease

Early-Stage Incorporation Strategy for Regioselective Labeling of Peptides using the 2-Cyanobenzothiazole/1,2-Aminothiol Bioorthogonal Click Reaction

Herein, we describe a synthetic strategy for the regioselective labeling of peptides by using a bioorthogonal click reaction between 2‐cyanobenzothiazole (CBT) and a 1,2‐aminothiol moiety. This methodology allows for the facile and site‐specific modification of peptides with various imaging agents, including fluorophores and radioisotope‐containing prosthetic groups. We investigated the feasibility of an early‐stage incorporation of dipeptide 1 into targeting vectors, such as c[RGDyK(C)] and HER2 pep, during solid‐phase peptide synthesis. Then, the utility of the click reaction to label bioactive peptides with a CBT‐modified imaging agent (FITC–CBT, 9) was assessed. The ligation reaction was found to be highly selective and efficient under various conditions. The fluorescently labeled peptides 2 and 3 were obtained in respective yields of 88 and 82 % under optimized conditions.We gratefully acknowledge the Leenaards Foundation (grant # 3699) and NSERC for financial support

Positron Emission Tomography Imaging of CD105 Expression with a 64Cu-Labeled Monoclonal Antibody: NOTA Is Superior to DOTA

Optimizing the in vivo stability of positron emission tomography (PET) tracers is of critical importance to cancer diagnosis. In the case of 64Cu-labeled monoclonal antibodies (mAb), in vivo behavior and biodistribution is critically dependent on the performance of the bifunctional chelator used to conjugate the mAb to the radiolabel. This study compared the in vivo characteristics of 64Cu-labeled TRC105 (a chimeric mAb that binds to both human and murine CD105), through two commonly used chelators: 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Flow cytometry analysis confirmed that chelator conjugation of TRC105 did not affect its CD105 binding affinity or specificity. PET imaging and biodistribution studies in 4T1 murine breast tumor-bearing mice revealed that 64Cu-NOTA-TRC105 exhibited better stability than 64Cu-DOTA-TRC105 in vivo, which resulted in significantly lower liver uptake without compromising the tumor targeting efficiency. In conclusion, this study confirmed that NOTA is a superior chelator to DOTA for PET imaging with 64Cu-labeled TRC105

Radiolabelled peptides for oncological diagnosis

Radiolabelled receptor-binding peptides targeting receptors (over)expressed on tumour cells are widely under investigation for tumour diagnosis and therapy. The concept of using radiolabelled receptor-binding peptides to target receptor-expressing tissues in vivo has stimulated a large body of research in nuclear medicine. The 111In-labelled somatostatin analogue octreotide (OctreoScan™) is the most successful radiopeptide for tumour imaging, and was the first to be approved for diagnostic use. Based on the success of these studies, other receptor-targeting peptides such as cholecystokinin/gastrin analogues, glucagon-like peptide-1, bombesin (BN), chemokine receptor CXCR4 targeting peptides, and RGD peptides are currently under development or undergoing clinical trials. In this review, we discuss some of these peptides and their analogues, with regard to their potential for radionuclide imaging of tumours

Glycomic analysis of life stages of the human parasite Schistosoma mansoni reveals developmental expression profiles of functional and antigenic glycan motifs

Contains fulltext : 155377.pdf (publisher's version ) (Open Access)Glycans present on glycoproteins and glycolipids of the major human parasite Schistosoma mansoni induce innate as well as adaptive immune responses in the host. To be able to study the molecular characteristics of schistosome infections it is therefore required to determine the expression profiles of glycans and antigenic glycan-motifs during a range of critical stages of the complex schistosome lifecycle. We performed a longitudinal profiling study covering schistosome glycosylation throughout worm- and egg-development using a mass spectrometry-based glycomics approach. Our study revealed that during worm development N-glycans with Galbeta1-4(Fucalpha1-3)GlcNAc (LeX) and core-xylose motifs were rapidly lost after cercariae to schistosomula transformation, whereas GalNAcbeta1-4GlcNAc (LDN)-motifs gradually became abundant and predominated in adult worms. LeX-motifs were present on glycolipids up to 2 weeks of schistosomula development, whereas glycolipids with mono- and multifucosylated LDN-motifs remained present up to the adult worm stage. In contrast, expression of complex O-glycans diminished to undetectable levels within days after transformation. During egg development, a rich diversity of N-glycans with fucosylated motifs was expressed, but with alpha3-core fucose and a high degree of multifucosylated antennae only in mature eggs and miracidia. N-glycan antennae were exclusively LDN-based in miracidia. O-glycans in the mature eggs were also diverse and contained LeX- and multifucosylated LDN, but none of these were associated with miracidia in which we detected only the Galbeta1-3(Galbeta1-6)GalNAc core glycan. Immature eggs also exhibited short O-glycan core structures only, suggesting that complex fucosylated O-glycans of schistosome eggs are derived primarily from glycoproteins produced by the subshell envelope in the developed egg. Lipid glycans with multifucosylated GlcNAc repeats were present throughout egg development, but with the longer highly fucosylated stretches enriched in mature eggs and miracidia. This global analysis of the developing schistosome's glycome provides new insights into how stage-specifically expressed glycans may contribute to different aspects of schistosome-host interactions