Keith Graham

Synthesis, 18F-Labeling, and in Vitro and in Vivo Studies of Bombesin Peptides Modified with Silicon-Based Building Blocks

The gastrin-releasing peptide receptor (GRPr) is overexpressed on various human tumors. The goal of our study was the synthesis of new 18F-labeled bombesin analogues for the PET imaging of GRPr expression in prostate tumor using a silicon-based one-step n. c. a. radiolabeling method. The silicon-containing building blocks were efficiently coupled to the N-terminus of the peptides via solid-phase synthesis. Radiolabeling of the obtained peptide precursors proceeded smoothly under acidic conditions (34-85% conversion). Using the di-tert-butyl silyl building block as labeling moiety, products containing a hydrolytically stable 18F-label were obtained. In in vitro receptor binding experiments 2-(4-(di-tert-butylfluorosilyl)phenyl)acetyl-Arg-Ava-Gln-Trp-Ala-Val-NMeGly-His-Sta-Leu-NH 2 ( 4b, IC50 = 22.9 nM) displayed a 12-fold higher binding affinity than 2-(4-(di-tert-butylfluorosilyl)phenyl)acetyl-Arg-Ava-Gln-Trp-Ala-Val-Gly-His(3Me)-Sta-Leu-NH2 ( 3b, IC50 = 276.6 nM), and 4b was therefore chosen for further evaluation. In vitro and ex vivo metabolite studies of [18F]4b showed no significant degradation. In biodistribution experiments, tumor uptake of [18F]4b was low and unspecific, whereas the GRPr-rich pancreas revealed a high and specific accumulation of the radiotracer. This study demonstrates the applicability of our silicon-based one-step n. c. a. radiolabeling method for the synthesis of new 18F-labeled bombesin derivatives. This innovative approach represents a general, straightforward access to radiolabeled peptides as PET imaging probes.

Direct One-Step18F-Labeling of Peptides via Nucleophilic Aromatic Substitution

Methods for the radiolabeling molecules of interest with [18F]-fluoride need to be rapid, convenient, and efficient. Numerous [18F]-labeled prosthetic groups, e.g., N-succinimidyl 4 [18F]-fluorobenzoate ([18F]-SFB), 4-azidophenacyl-[18F]-fluoride ([18F]-APF), and 1-(3-(2-[18F]fluoropyridin-3-yloxy)propyl)pyrrole-2,5-dione ([18F]-FpyMe), for conjugating to biomolecules have been developed. As the synthesis of these prosthetic groups usually requires multistep procedures, there is still a need for direct methods for the nucleophilic [18F]-fluorination of biomolecules. We report here on the development of a procedure based on the trimethylammonium (TMA) leaving group attached to an aromatic ring and activated with different electron-withdrawing groups (EWGs). A series of model compounds containing different electron-withdrawing substituents, a trimethylammonium leaving group, and carboxylic functionality for subsequent coupling to peptides were designed and synthesized. The optimal model compound, 2-cyano-4-(methoxycarbonyl)-N,N,N-trimethylbenzenaminium trifluoromethanesulfonate, was converted to carboxylic acid and coupled to peptides. The results of the one-step [18F]-fluorination of tetrapeptides and bombesin peptides show that the direct 18F-labeling of peptides is feasible under mild conditions and in good radiochemical yields.

18F-Labeled Bombesin Analog for Specific and Effective Targeting of Prostate Tumors Expressing Gastrin-Releasing Peptide Receptors

Bombesin is a peptide exhibiting high affinity for the gastrin-releasing peptide receptor (GRPr), which is highly overexpressed on prostate cancer cells. In the present study, we developed an 18F-labeled bombesin analog, 18F-BAY 86-4367, which is currently being clinically tested for use in PET of prostate cancer. Methods: In vitro pharmacologic studies were performed to characterize the nonradioactive (19F) standard of the bombesin analog for binding affinity and selectivity for GRPr. The stability of 18F-BAY 86-4367 was determined in murine and human plasma. In vivo, the tumor-targeting potential and pharmacokinetic profile of the 18F tracer were analyzed with biodistribution experiments and PET studies of prostate tumor–bearing mice. Results: The nonradioactive (19F) standard of the bombesin analog showed subnanomolar and GRPr-selective binding affinity. The stability of the tracer in murine and human plasma was found to be high. In 2 prostate cancer xenograft models (PC-3 and LNCaP), 18F-BAY 86-4367 showed more specific and effective GRPr-based targeting in vivo than the benchmark radiotracers 18F-fluoroethylcholine and 18F-FDG. In addition, rapid tumor targeting and fast renal excretion (∼70%) and hepatobiliary excretion (∼10%) were identified in both xenograft models. Furthermore, PET studies provided clear and specific visualization of PC-3 tumors in mice. Conclusion: Favorable preclinical data showing specific and effective tumor targeting by 18F-BAY 86-4367 suggest that a clinical trial be undertaken to test its diagnostic utility in PET for prostate carcinoma patients.

Preclinical Evaluation of the Breast Cancer Cell-Binding Peptide, p160

Purpose: Selective delivery of drugs into the target tissue is expected to result in high drug concentrations in the tissue of interest and therefore enhanced drug efficacy. To develop a peptide-based radiopharmaceutical, we investigated the properties of a peptide with affinity for human breast cancer, which has been selected through phage display. Experimental Design: The bioactivity of the p160 peptide (VPWMEPAYQRFL) was evaluated in vitro and in vivo. The specific binding to human breast cancer MDA-MB-435 cells was confirmed in competition experiments. Internalization of the peptide was investigated with confocal microscopy. Furthermore, the biodistribution of 131I-labeled p160 was studied in tumor-bearing mice. In vivo stability was evaluated at different periods after tracer administration using high-performance liquid chromatography analysis. Results: The binding of 125I-labeled p160 was inhibited up to 95% by the unlabeled peptide with an IC50 value of 0.6 μmol/L. In addition, 40% of the total bound activity was found to be internalized into the human breast cancer cells. Although a rapid degradation was seen, biodistribution studies in nude mice showed a higher uptake in tumor than in most of the organs. Perfusion of the animals caused a reduction of the radioligand accumulation in the healthy tissues, whereas the tumor uptake remained constant. A comparison of [131I]p160 with a 131I-labeled Arg-Gly-Asp peptide revealed a higher tumor-to-organ ratio for [131I]p160. Conclusions: p160 has properties that make it an attractive carrier for tumor imaging and the intracellular delivery of isotopes or chemotherapeutic drugs.

4-[18F]fluoroglutamic acid (BAY 85-8050), a new amino acid radiotracer for PET imaging of tumors: synthesis and in vitro characterization.

There is a high demand for tumor specific PET tracers in oncology imaging. Besides glucose, certain amino acids also serve as energy sources and anabolic precursors for tumors. Therefore, (18)F-labeled amino acids are interesting probes for tumor specific PET imaging. As glutamine and glutamate play a key role in the adapted intermediary metabolism of tumors, the radiosynthesis of 4-[(18)F]fluoro l-glutamic acid (BAY 85-8050) as a new specific PET tracer was established. Cell-uptake studies revealed specific tumor cell accumulation.

In vitro and in vivo characterization of novel 18F-labeled bombesin analogues for targeting GRPR-positive tumors.

The gastrin-releasing peptide receptor (GRPR) is overexpressed on a number of human tumors and has been targeted with radiolabeled bombesin analogues for the diagnosis and therapy of these cancers. Seven bombesin analogues containing various linkers and peptide sequences were designed, synthesized, radiolabeled with (18)F, and characterized in vitro and in vivo as potential PET imaging agents. Binding studies displayed nanomolar binding affinities toward human GRPR for all synthesized bombesin analogues. Two high-affinity peptide candidates 6b (K(i) = 0.7 nM) and 7b (K(i) = 0.1 nM) were chosen for further in vivo evaluation. Both tracers revealed specific uptake in GRPR-expressing PC-3 tumors and the pancreas. Compared to [(18)F]6b, compound [(18)F]7b was characterized by superior tumor uptake, higher specificity of tracer uptake, and more favorable tumor-to-nontarget ratios. In vivo PET imaging allowed for the visualization of PC-3 tumor in nude mice suggesting that [(18)F]7b is a promising PET tracer candidate for the diagnosis of GRPR-positive tumors in humans.

Radiofluorinated derivatives of 2-(phosphonomethyl)pentanedioic acid as inhibitors of prostate specific membrane antigen (PSMA) for the imaging of prostate cancer.

For prostate cancer, prostate specific membrane antigen (PSMA) has been identified as a diagnostic and therapeutic target. Fluorinated derivatives of 2-(phosphonomethyl)pentanedioic acid were designed and synthesized to explore whether this fluorine-substituent is tolerated in the pentanedioic acid moiety that is common to almost all PSMA targeting small molecule inhibitors. The binding affinities of the racemic and individual stereoisomers of 2-fluoro-4-(phosphonomethyl)pentanedioic acid were determined and showed that the introduction of fluorine was well tolerated. The radiosynthesis of the analogous 2-[(18)F]fluoro-4-(phosphonomethyl)pentanedioic acid was developed and evaluated in vivo with the PSMA positive LNCaP human prostate cancer cell. The biological results demonstrated specific binding of the tracer to PSMA positive tumors in mice. These results warrant the further evaluation of this class of compounds as radiolabeled tracers for the detection and staging of prostate cancer.

Pharmacological properties of hydrophilic and lipophilic derivatives of octreotate

Derivatives of somatostatin (SST) represent the most important peptides for receptor targeting in oncological applications. Whereas the pharmacophor in somatostatin receptor-affine substances has been thoroughly investigated, the influence of modifications at the N-terminal has not yet been systematically studied. In order to investigate the influence of hydrophilic versus lipophilic modifications at the N-terminal end, a series of homologous derivatives of Tyr3-octreotate modified with oligomers of ethylene glycol or fatty acids were synthesized. For this purpose, Tyr3-octreotate was assembled using solid phase peptide synthesis and the fatty acids or oligomers of ethylene glycol were conjugated to the N-terminal end. The oligomers of ethylene glycol were activated by 4-nitrophenylchloroformate to obtain carbamate-linked hydrophilic compounds. The receptor affinities of these compounds were determined by competition experiments with [125I]Tyr3-octreotide on rat cortex membranes. The hydrophilic derivatives and the short chain lipophilic derivatives revealed IC50 values between 0.66 +/- 0.02 nM and 2.16 +/- 0.31 nM respectively. After labeling with (125)I the organ distribution of selected derivatives was investigated in Lewis rats bearing the rat pancreatic tumor CA20948. All of the compounds showed high tumor uptake. The peptides conjugated to oligomers of ethylene glycol showed low uptake into the liver and kidneys. Increasing the length of the fatty acids resulted in a remarkable decrease in kidney uptake. In conclusion, the systematic modifications at the N-terminal result in a low effect on the receptor affinity but allow the modulation of the pharmacokinetic properties of octreotide derivatives.

A general method for functionalising both the C- and N-terminals of Tyr3-octreotate

Abstract A new method has been developed for solid-phase peptide synthesis (SPPS) via the Fmoc strategy that allows functionalisation of both the C- and N-terminals of Tyr 3 -octreotate. N -α-Fluorenylmethyloxycarbonyl- l -threonine allyl ester, the starting amino acid, was loaded onto the 3,4-dihydro-2 H -pyran-2-ylmethoxymethyl polystyrene (DHP) resin via its side chain alcohol group and the cyclic Tyr 3 -octreotate was synthesised. The C- and N-terminals were functionalised with DOTA (1,4,7,10-tetraazacyclodecane- N ′, N ′′, N ′′′, N ′′′′-tetraacetic acid) and HYNIC (6-hydrazinonicotinic acid).

Fluorescent Somatostatin Receptor Probes for the Intraoperative Detection of Tumor Tissue with Long-Wavelength Visible Light

Targeted fluorescent dyes are of substantial value for the intraoperative delineation of primary tumors and metastatic lesions. For this purpose long-wavelength red light (lambda=550-650 nm) offers advantages because of good tissue penetration and direct visibility. Since somatostatin receptors (SSTR) are overexpressed in a number of tumors, a series of potentially tumor-selective peptide-dye conjugates were synthesized by solid-phase peptide synthesis (SPPS). The octapeptides octreotate, Tyr(3)-octreotate and Tyr(3)-octreotide were employed and exhibited high affinity for somatostatin receptors (SSTR). The fluorescent dyes rhodamine 101, sulforhodamine B acid chloride, sulforhodamine 101 or rhodamine B isothiocyanate were conjugated either directly or via spacers, for example the peptidase-labile pentapeptide sequence Ala-Leu-Ala-Leu-Ala. The conjugates were completely assembled on the solid support: Fmoc-SPPS, cyclization via a disulfide linkage, N-terminal attachment of a spacer, and linkage to the fluorescent dye. An in vitro competition assay revealed that the conjugates bind to SSTRs with IC(50) values between 0.7 and 89 nM. The conjugates were generally stable to hydrolysis at pH 7-8 in buffer or serum. However, the rhodamine 101 conjugates revealed a loss of absorption at alkaline pH due to conversion to a neutral spirolactam form, as characterized by NMR.