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.

Novel DOTA-neurotensin analogues for 111In scintigraphy and 68Ga PET imaging of neurotensin receptor-positive tumors.

Overexpression of the high affinity neurotensin receptor 1 (NTSR1), demonstrated in several human cancers, has been proposed as a new marker for human ductal pancreatic carcinoma and as an independent factor for poor prognosis for ductal breast cancer, head and neck squamous cell carcinoma, and non-small cell lung cancer. The aim of the present study was to develop new DOTA-neurotensin analogues for positron emission tomography (PET) imaging with (68)Ga and for targeted radiotherapy with (90)Y or (177)Lu. We synthesized a DOTA-neurotensin analogue series. Two of these peptides bear two sequence modifications for metabolic stability: DOTA-NT-20.3 shares the same peptide sequence as the previously described DTPA-NT-20.3. In the sequence of DOTA-NT-20.4, the Arg(8)-Arg(9) bond was N-methylated instead of the Pro(7)-Arg(8) bond in DOTA-NT-20.3. An additional sequence modification was introduced in DOTA-LB119 to increase stability. A spacer was added between DOTA and the peptide sequence to increase affinity. Binding to HT29 cells, which express NTSR1, in vivo stability, and biodistribution of the various analogues were compared, and the best candidate was used to image tumors of various sizes with the microPET in mice. (111)In-DOTA-NT-20.3, in spite of a relatively high uptake in kidneys, showed specific tumor uptake and elevated tumor to other organ uptake ratios. High contrast images were obtained at early time points after injection that allowed tumor detection at a time interval postinjection appropriate for imaging with the short-lived radionuclide (68)Ga. (111)In-DOTA-NT-20.4 displayed inferior binding to HT29 cells and reduced tumor uptake. (111)In-DOTA-LB119 displayed at early time points a significantly lower renal uptake but also a lower tumor uptake than (111)In-DOTA-NT-20.3, although binding to HT29 cells was similar. (68)Ga-DOTA-NT-20.3 displayed higher tumor uptake than (68)Ga-DOTA-LB119 and allowed the detection of very small tumors by PET. In conclusion, DOTA-NT-20.3 is a promising candidate for (68)Ga-PET imaging of neurotensin receptor-positive tumors. DOTA-NT-20.3 may also be considered for therapy, as the yttrium-labeled peptide has higher affinity than that of the indium-labeled one. A prerequisite for therapeutic application of this neurotensin analogue would be to lower kidney uptake, for example, by infusion of basic amino acids, gelofusin, or albumin fragments, to prevent nephrotoxicity, as with radiolabeled somatostatin analogues.

Molecular assembly of multifunctional 99(m)Tc radiopharmaceuticals using "clickable" amino acid derivatives.

Synthetic strategies that enable the efficient and selective combination of different biologically active entities hold great promise for the development of multifunctional hybrid conjugates useful for biochemical and medical applications. Starting from side-chain-functionalized N(α)-propargyl lysine derivatives, conjugates containing a 99(m)Tc-based imaging probe for SPECT and two different moieties (e.g., tumor-targeting vectors, pharmacological modifiers, affinity tags, or second imaging probes) can be assembled using the Cu(I)-catalyzed alkyne-azide cycloaddition in efficient one-pot protocols. This strategy was successfully applied to the preparation of a 99(m)Tc-labeled conjugate comprising a tumor-targeting peptide sequence (bombesin(7-14)) and a low-molecular-weight albumin binder, a pharmacological modifier that prolongs the blood circulation time of the conjugate. Evaluation of the conjugate in vitro and in vivo provided promising results for its use as an imaging agent for the visualization of tumors positive for the gastrin-releasing peptide receptor. The methodology presented herein provides an attractive synthetic tool for the preparation of multifunctional 99(m)Tc-based radiopharmaceuticals with significant potential for a multitude of applications.

Synthesis and evaluation of bombesin analogues conjugated to two different triazolyl-derived chelators for (99m)Tc labeling.

Overexpression of the gastrin-releasing peptide receptor (GRPR) in a variety of human carcinomas has provided a means of diagnosis and treatment. Previously we reported a metabolically stable (N(α)His)Ac-ßAla-ßAla-[Cha(13),Nle(14)]BBS(7-14) analogue with high affinity for the GRPR. We have also shown that the biodistribution pattern of this fairly lipophilic, radiolabeled peptide can be enhanced by glycation, which is easily carried out by Cu(I)-catalyzed cycloaddition. Herein, we further elaborate this "click approach" in the synthesis of a new series of triazole-based chelating systems as alternatives to the (N(α)His)Ac chelator for labeling with the (99m)Tc(CO)(3) core. The bombesin analogues, containing these new chelating systems, were evaluated with regard to their synthesis and in vitro and in vivo properties, and were compared with their (N(α)His)Ac counterparts. The influence of the chelator on biodistribution properties was less than that of glycation, which clearly improved the tumor-to-background ratios.

A click approach to structurally diverse conjugates containing a central di-1,2,3-triazole metal chelate.

The selective and efficient synthesis of novel tridentate metal chelating systems containing two 1,4-disubstituted 1,2,3-triazole heterocycles obtained via the copper(I)-catalyzed cycloaddition of alkynes and azides (click reaction) is described. The constructs are shown to be efficient ligand systems for the chelation of fac-[M(CO)(3)(H(2)O)(3)](+) (M=(99m)Tc, Re) yielding well- defined and stable complexes. The organometallic (99m)Tc conjugates are suitable for application as diagnostic radiotracers for single photon emission computed tomography (SPECT) as demonstrated in vivo with a fragment of the tumor-targeting bombesin peptide functionalized with a di-1,2,3-triazole chelator and radiolabeled with [(99m)Tc(CO)(3)](+). Starting from readily available dialkyne precursors, the central chelating systems are formed as the conjugates are assembled by click reaction with azide-functionalized entities. Depending on the nature of the azide substrates employed (e.g. lipophilic or hydrophilic residues) pharmacologically relevant characteristics of the final metal conjugate such as hydrophilicity or overall charge can be readily modulated. The procedures described also enable the facile introduction of other probes into the metal conjugate, providing access to potential multimodal imaging agents.

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.

"Click to chelate": synthesis and installation of metal chelates into biomolecules in a single step.

Click chemistry has been employed for the assembly of novel and efficient triazole-based multidentate chelating systems while simultaneously attaching them to molecules of biological interest. The "click-to-chelate" approach offers a powerful new tool for the modification of (bio)molecules with metal chelators for potential diagnostic and therapeutic applications.