Luc Brans

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.

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

Assemble & chelate: Click chemistry enables the efficient and selective synthesis of structurally diverse conjugates containing a central di‐1,2,3‐triazole chelator for complexation with [99mTc(CO)3]+. Use of appropriate building blocks allows the modulation of pharmacological relevant characteristics of the conjugate, or the introduction of secondary probes suitable for imaging modalities other than single photon emission computed tomography (SPECT).

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.

PEGylation of 99mTc-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 β(3)hLys-βAla-βAla 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.

Molecular Assembly of Multifunctional 99mTc 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 99mTc‐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 CuI‐catalyzed alkyne–azide cycloaddition in efficient one‐pot protocols. This strategy was successfully applied to the preparation of a 99mTc‐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 99mTc‐based radiopharmaceuticals with significant potential for a multitude of applications.

Glycation Methods for Bombesin Analogs Containing the (NαHis)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 99mTc‐radiolabeled bombesin ([Cha13, Nle14]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 [Cha13, Nle14]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 α‐d‐glucose to an amino‐oxyacetylated [Cha13, Nle14]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 N3‐β‐d‐glucose to a propargylglycine‐containing [Cha13, Nle14]BBS(7–14) analog, using a catalytic amount of Cu(I)I. All glycated [Cha13, Nle14]BBS(7–14) analogs showed high affinity for the gastrin‐releasing peptide receptor and rapid accumulation into PC‐3 tumor cells.

Synthesis and Evaluation of Bombesin Analogues Conjugated to Two Different Triazolyl-Derived Chelators for 99mTc 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 (NαHis)Ac‐βAla‐βAla‐[Cha13,Nle14]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 CuI‐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 (NαHis)Ac chelator for labeling with the 99mTc(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 (NαHis)Ac counterparts. The influence of the chelator on biodistribution properties was less than that of glycation, which clearly improved the tumor‐to‐background ratios.

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

BackgroundRadiolabelled 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.MethodsDerivatisation 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 β3hLys-βAla-βAla 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.ResultsThe 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).ConclusionsPEGylation 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.