Influence of the Molecular Charge on the Biodistribution of Bombesin Analogues Labeled with the [^99mTc(CO)₃]-Core

Abstract

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.

Influence of Labelling Methods on Biodistribution and Imaging Properties of Radiolabelled Peptides for Visualisation of Molecular Therapeutic Targets

“…Stability of such linker to peptidases should be taken into account, as it has been shown by García Garayoa and coworkers [123][124][125] in a series of studies on bombesin analogues labelled with 99m Tc(CO) 3 using retro[Ncarboxymethyl-histidine]. Blood plasma stability of radiopeptides with three linkers: 3,6-dioxa-8-aminooctanoic acid, Ala-Ala and Lys(sha)-Ala-Ala (sha = shikimic acid) and a conjugate without linker were compared.…”

“…The chemical nature of the linker may also influence its effect on the affinity. Three linkers containing 3 hGlu and Ala ( 3 hGluAla-Ala, 3 hGlu-3 hGlu-Ala, and 3 hGlu-3 hGlu-3 hGlu) were investigated in order to increase the hydrophilicity of 99m Tc(CO) 3 -retro[N -carboxymethyl-histidine]-BBN (7)(8)(9)(10)(11)(12)(13)(14) conjugates [125]. It was found that the introduction of two charged amino acids increased the hydrophilicity while keeping the affinity, but the affinity was decreased by a linker containing only charged amino acids.…”

“…This effect was clearly pronounced for 99m Tc-labelled bombesin. It has been shown that positively charged linkers between the bombesin derivative and the 99m Tc(CO) 3 -retro[N -carboxymethyl-histidine] favour internalisation while linkers with more than one negative charge would have an adverse influence [125] . Alves et al [165] studied technetium labeled BBN [7][8][9][10][11][12][13][14] conjugates labelled with [ 99m Tc(CO) 3 ] + using a pyrazolyl chelate attached via -alanine, serylserylserine (SSS) or glycylglycylclgycine (GGG) linkers.…”

Molecular Assembly of Multifunctional 99mTc Radiopharmaceuticals Using “Clickable” Amino Acid Derivatives

“…Tc-labeled derivatives of bombesin (BBS), [22,33] a regulatory peptide that targets the gastrin-releasing peptide (GRP) receptor, which is overexpressed in various tumor types such as prostate, breast, and small-cell lung cancer. [34] Like other radiolabeled regulatory peptides potentially useful as imaging probes for the detection of tumors and metastasis, [35] radiotracers derived from BBS or its binding sequence BBS (7)(8)(9)(10)(11)(12)(13)(14) are rapidly degraded both extracellularly and intracellularly by peptidases.…”

“…[12,33] Alkyne 17 equipped with the albumin binding molecule C (Scheme 3) was conjugated to azidopeptide 23 by CuAAC in a mixture of water and ethanol at 60 8C to yield BBS(7-14) derivative L24. Reference compound N a HisAc-BBS (7)(8)(9)(10)(11)(12)(13)(14), identical in all respects but lacking the albumin binding moiety and functionalized with the established N(a)-HisAc chelating system, was prepared by SPPS.…”