PEGylation, increasing specific activity and multiple dosing as strategies to improve the risk-benefit profile of targeted radionuclide therapy with 177Lu-DOTA-bombesin analogues.

BACKGROUND: Radiolabelled bombesin (BN) conjugates are promising radiotracers for imaging and therapy of breast and prostate tumours, in which BN2/gastrin-releasing peptide receptors are overexpressed. We describe the influence of the specific activity of a 177Lu-DOTA-PEG5k-Lys-B analogue on its therapeutic efficacy and compare it with its non-PEGylated counterpart. METHODS: Derivatisation of a stabilised DOTA-BN(7-14)[Cha13,Nle14] analogue with a linear PEG molecule of 5 kDa (PEG5k) was performed by PEGylation of the ϵ-amino group of a ß3hLys-ßAla-ßAla spacer between the BN sequence and the DOTA chelator. The non-PEGylated and the PEGylated analogues were radiolabelled with 177Lu. In vitro evaluation was performed in human prostate carcinoma PC-3 cells, and in vivo studies were carried out in nude mice bearing PC-3 tumour xenografts. Different specific activities of the PEGylated BN analogue and various dose regimens were evaluated concerning their therapeutic efficacy. RESULTS: The specificity and the binding affinity of the BN analogue for BN2/GRP receptors were only slightly reduced by PEGylation. In vitro binding kinetics of the PEGylated analogue was slower since steady-state condition was reached after 4 h. PEGylation improved the stability of BN conjugate in vitro in human plasma by a factor of 5.6. The non-PEGylated BN analogue showed favourable pharmacokinetics already, i.e. fast blood clearance and renal excretion, but PEGylation improved the in vivo behaviour further. One hour after injection, the tumour uptake of the PEG5k-BN derivative was higher compared with that of the non-PEGylated analogue (3.43 ± 0.63% vs. 1.88 ± 0.4% ID/g). Moreover, the increased tumour retention resulted in a twofold higher tumour accumulation at 24 h p.i., and increased tumour-to-non-target ratios (tumour-to-kidney, 0.6 vs. 0.4; tumour-to-liver, 8.8 vs. 5.9, 24 h p.i.). In the therapy study, both 177Lu-labelled BN analogues significantly inhibited tumour growth. The therapeutic efficacy was highest for the PEGylated derivative of high specific activity administered in two fractions (2 × 20 MBq = 40 MBq) at day 0 and day 7 (73% tumour growth inhibition, 3 weeks after therapy). CONCLUSIONS: PEGylation and increasing the specific activity enhance the pharmacokinetic properties of a 177Lu-labelled BN-based radiopharmaceutical and provide a protocol for targeted radionuclide therapy with a beneficial anti-tumour effectiveness and a favourable risk-profile at the same time.

PEGylation of (99m)Tc-labeled bombesin analogues improves their pharmacokinetic properties.

INTRODUCTION: Radiolabeled bombesin (BN) conjugates are promising radiotracers for imaging and therapy of breast and prostate tumors in which BN(2)/gastrin-releasing peptide (GRP) receptors are overexpressed. However, the low in vivo stability of BN conjugates may limit their clinical application. In an attempt to improve their pharmacokinetics and counteract their rapid enzymatic degradation, we prepared a series of polyethylene glycol (PEG)-ylated BN(7-14) analogues for radiolabeling with (99m)Tc(CO)(3) and evaluated them in vitro and in vivo. METHODS: Derivatization of a stabilized (N(α)His)Ac-BN(7-14)[Cha(13),Nle(14)] analogue with linear PEG molecules of various sizes [5 kDa (PEG(5)), 10 kDa (PEG(10)) and 20 kDa (PEG(20))] was performed by PEGylation of the ɛ-amino group of a ß(3)hLys-ßAla-ßAla spacer between the stabilized BN sequence and the (N(α)His)Ac chelator. The analogues were then radiolabeled by employing the (99m)Tc-tricarbonyl technique. Binding affinity and internalization/externalization studies were performed in vitro in human prostate carcinoma PC-3 cells. Stability was investigated in vitro in human plasma and in vivo in Balb/c mice. Finally, single photon emission computed tomography (SPECT)/X-ray computed tomography studies were performed in nude mice bearing PC-3 tumor xenografts. RESULTS: PEGylation did not affect the binding affinity of BN analogues, as the binding affinity for BN(2)/GRP receptors remained high (K(d)<0.9 nM). However, in vitro binding kinetics of the PEGylated analogues were slower. Steady-state condition was reached after 4 h, and the total cell binding was 10 times lower than that for the non-PEGylated counterpart. Besides, PEGylation improved the stability of BN conjugates in vitro and in vivo. The BN derivative conjugated with a PEG(5) molecule showed the best pharmacokinetics in vivo, i.e., faster blood clearance and preferential renal excretion. The tumor uptake of the (99m)Tc-PEG(5)-Lys-BN conjugate was slightly higher compared to that of the non-PEGylated analogue (3.91%±0.44% vs. 2.80%±0.28% injected dose per gram 1 h postinjection, p.i.). Tumor retention was also increased, resulting in a threefold higher amount of radioactivity in the tumor at 24 h p.i. Furthermore, decreased hepatobiliary excretion and increased tumor-to-nontarget ratios (tumor-to-blood: 17.1 vs. 2.1; tumor-to-kidney: 1.1 vs. 0.4; tumor-to-liver: 5.8 vs. 1.0, 24 h p.i.) were observed and further confirmed via small-animal SPECT images 1 h p.i. CONCLUSION: PEGylation proved to be an effective strategy to enhance the tumor-targeting potential of (99m)Tc-labeled BN-based radiopharmaceuticals and probably other radiolabeled peptides.

Bis(thiosemicarbazones) as bifunctional chelators for the room temperature 64-copper labeling of peptides.

A range of new carboxylate functionalised bis(thiosemicarbazone) ligands and their Cu(II) complexes have been prepared, fully characterised and radiolabeled in high yield with both (64)Cu and (99m)Tc. Conjugation to a bombesin derivative was achieved using standard solid phase synthetic methodologies and the (64)Cu-labeled conjugate was shown to have good tumour uptake in mice with xenografted PC-3 tumours.

The use of time-averaged 3JHH restrained molecular dynamics (tar-MD) simulations for the conformational analysis of five-membered ring systems: methodology and applications.

Because of its presence in many molecules of biological relevance, the conformational analysis of five-membered rings using (3)J(HH) scalar coupling data from NMR is a topic of considerable interest. Typically, conformational analysis involves the use of a well-established mathematical procedure, originally developed by de Leeuw et al., that fits two rigid conformations to the available experimental data. This so-called pseudorotation analysis approach is not without problems, however, as chemically unrealistic conformations are sometimes generated from the data. Here, we present our investigations in the use of time-averaged restrained molecular dynamics simulations as a generic tool to determine the conformations that agree with experimental (3)J(HH) scalar coupling data. For this purpose, a set of six ribose-based molecules has been used as model compounds. The influence of several modeling parameters is assessed and optimized values are proposed. The results obtained with the tar-MD approach are compared to those obtained from the two conformer fitting procedure. Interpretation of the latter is facilitated by the introduction of a fitting error analysis that allows mapping the solution space of the fitting procedure. The relative merits of both methods and the advantages that result from the use of a force field and a time-averaged restraint potential for the experimental data are discussed. When combined, both techniques allow an enhanced understanding of the molecules' conformational behavior and prevent possible overinterpretation. In view of the very reasonable computational burden of a tar-MD simulation for the systems investigated here, the approach should be generally applicable.

Novel glycated [99mTc(CO)3]-labeled bombesin analogues for improved targeting of gastrin-releasing peptide receptor-positive tumors.

Radiolabeled bombesin (BBS) analogues are promising pharmaceuticals for imaging of cancer cells expressing gastrin-releasing peptide receptors (GRPR). However, most of the radiolabeled BBS derivatives show a high accumulation of activity in the liver and a strong hepatobiliary excretion, both unfavorable for imaging and therapy of abdominal lesions. For this reason, we introduced hydrophilic carbohydrated linker moieties into our BBS analogues to reduce the abdominal accumulation and to improve the tumor-to-background ratios. A stabilized BBS(7-14) sequence bearing the (NalphaHis)Ac-chelator was modified with amino acid linkers containing a lysine or propargylglycine residue. The epsilon-amino group of a lysine was either coupled to shikimic acid or reacted with glucose to form the Amadori conjugate. Alternatively, a glucose was attached to the peptide via "click" chemistry with the propargylglycine side chain. The peptides were synthesized on Rink amide resin using solid-phase peptide synthesis and labeled with 99mTc using the tricarbonyl technique. Binding and degradation were tested in Vitro in GRPR-expressing PC-3 cells. Biodistribution and SPECT/CT imaging studies were performed in nude mice bearing PC-3 tumor xenografts. The new peptides showed a log D between -0.2 and -0.5 and kept the high affinity for GRPR with Kd values of <0.5 nM. In Vitro, they were rapidly internalized into the tumor cells and showed an increased cellular retention and stability (t(1/2 )>35 min). In ViVo, all new compounds exhibited higher tumor-to-background ratios compared to the nonglycated reference. Thus, the best results were obtained with the triazole coupled glucose with a 4-fold increased uptake and retention in tumor tissue (3.6 and 2.5%ID/g at 1.5 h and 5 h p.i, respectively) and a significantly reduced accumulation in the liver (0.6 vs 2.4%ID/g, 1.5 h p.i., respectively). Apart from higher tumor-to-liver ratios (17-fold, 1.5 h p.i.), both tumor-to-kidney and tumor-to-blood ratios could be significantly improved by a factor of 1.5 and 2.7, respectively (1.5 h p.i., P<0.05). The imaging studies proved the reduction of abdominal background, and tumor xenografts could clearly be visualized. In conclusion, the introduction of a carbohydrated linker substantially improved the biodistribution properties of BBS analogues labeled with the 99mTc-tricarbonyl core.

Glycation methods for bombesin analogs containing the (NalphaHis)Ac chelator for 99mTc(CO)3 radiolabeling.

The overexpression of peptide receptors in a variety of human carcinomas has generated considerable interest in peptide-based radiopharmaceuticals for peptide receptor imaging and peptide receptor radiotherapy. The gastrin-releasing peptide receptor is overexpressed in human prostate-, breast-, colon- and small cell lung carcinoma cells. We have developed metabolically stable (99m)Tc-radiolabeled bombesin ([Cha(13), Nle(14)]BBS(7-14)) analogs, which bind with high affinity to the gastrin-releasing peptide receptors. However, because of their lipophilicity, they showed unfavorable biodistribution with high hepatic accumulation and hepatobiliary excretion. We now report a study of different glycation methods for [Cha(13), Nle(14)]BBS(7-14) analogs to improve their biodistribution profile. Whereas the glycation using the Maillard reaction was problematic, resulting in low yields, selective introduction of the glycomimetic shikimic acid to the side chain of a Lys residue was possible. A chemoselective ligation of alpha-D-glucose to an amino-oxyacetylated [Cha(13), Nle(14)]BBS(7-14) analog could be achieved, but was complicated by the co-elution of starting peptide and glycopeptide. The best procedure consisted of the [1,3]-cycloaddition of N(3)-beta-D-glucose to a propargylglycine-containing [Cha(13), Nle(14)]BBS(7-14) analog, using a catalytic amount of Cu(I)I. All glycated [Cha(13), Nle(14)]BBS(7-14) analogs showed high affinity for the gastrin-releasing peptide receptor and rapid accumulation into PC-3 tumor cells.

Influence of the molecular charge on the biodistribution of bombesin analogues labeled with the [99mTc(CO)3]-core.

The overexpression of Bombesin (BBS) receptors on a variety of human cancers make them interesting targets for tumor imaging and therapy. Analogues of the neuropeptide BBS have been functionalized with the (NalphaHis)- chelator for labeling with the 99mTc-tricarbonyl core. The introduction of a betaAla-betaAla linker between the stabilized BBS binding sequence and the chelator led to increased tumor uptake but still rather unfavorable in ViVo properties. Novel polar linkers, with different charge, have been introduced in the molecule and tested for their influence on the biodistribution. The new analogues showed a shift in hydrophilicity from a Log D=0.9 to Log D values between 0.4 and -2.2. All compounds kept the increased stability in both human plasma (t(1/2)>16 h) and in tumor cells (t(1/2)=30-40 min). The compounds with Log D values between +1 and -1 showed the highest binding affinities with Kd values of <0.5 nM, as well as the highest cellular uptake. However, higher hydrophilicity (Log D < -1.8) led to lower affinity and a substantial decrease of internalization. The introduction of a positive charge (beta3hLys) resulted in unfavorable biodistribution, with increased kidney uptake. The introduction of an uncharged hydroxyl group (beta3hSer) improved the biodistribution, resulting in significantly better tumor-to-tissue ratios. The compound with one single negative charge (beta3hGlu) showed a significant increase in the tumor uptake (2.1+/-0.6% vs 0.80+/-0.35% ID/g in comparison to the betaAla-betaAla analogue) and also significantly higher tumor-to-tissue ratios. The specificity of the in ViVo uptake was confirmed by coinjection with natural BBS. Moreover, the analogue provided a much clearer image of the tumor xenografts in the SPECT/CT studies. The introduction of a single negative charge may be useful in the development of new BBS analogues to obtain an improved biodistribution profile, with increased tumor uptake and better imaging.

Chemical and biological characterization of new Re(CO)3/[99mTc](CO)3 bombesin analogues.

INTRODUCTION: Bombesin, a neuropeptide with potential for breast and prostate tumor targeting, is rapidly metabolized in vivo, and as a result, uptake in tumor xenografts in mice is poor. An improvement can be expected from the introduction of nonnatural amino acids and spacers. Leu13 was replaced by cyclohexylalanine and Met14 by norleucine. Two spacers, -betaAla-betaAla- and 3,6-dioxa-8-aminooctanoic acid, were inserted between the receptor-binding amino acid sequence (7-14) of bombesin (BBS) and the retroN(alpha)-carboxymethyl histidine chelator used for labeling with the [99mTc](CO)3 core and the rhenium (Re) congener. METHODS: The biological characterization of the new compounds was performed both in vitro on prostate carcinoma PC-3 cells (binding affinity, internalization/externalization) and in vivo (biodistribution in nude mice with tumor xenografts). The stability was also investigated in human plasma. The Re analogues were prepared for chemical characterization. RESULTS: The nonnatural amino acids led to markedly slower degradation in human plasma and PC-3 cell cultures. The receptor affinity of the new technetium 99m ([99mTc])-labeled BBS analogues was similar to the unmodified compound with Kd<1 nM. Uptake in the pancreas and in PC-3 tumor xenografts in nude mice was blocked by unlabeled BBS. The best target-to-nontarget uptake ratio was clearly due to the presence of the more polar spacer, -betaAla-betaAla-. CONCLUSIONS: The different spacers did not have a significant effect on stability or receptor affinity but had a clear influence on the uptake in healthy organs and tumors. Uptake in the kidneys was lower than in the liver, which is likely to be due to the lipophilicity of the compounds. A specific, high uptake was also observed in the gastrin-releasing peptide receptor-rich pancreas. Thus, with the introduction of spacers the in vivo properties of the compounds can be improved while leaving the affinity unaffected.