One goal of this investigation was to develop a polymer conjugated with multiple copies of peptide nucleic acid (PNA) and with pharmacokinetic properties suitable for applications in vivo. The second goal was to establish whether the multiple copies of PNA on the polymer could be targeted by hybridization in vitro and in vivo with (99m)Tc-labeled complementary PNA (cPNA). If successful, this approach could then be considered in further investigations as an alternative to existing pretargeting approaches because of the potential for signal amplification in the target. A 80 KDa poly(methyl vinyl ether-alt-maleic acid) (PA) polymer was conjugated with multiple copies of PNA and with multiple copies of poly(ethylene glycol) (PEG) by reacting the NHS derivative of PA with the amine derivatives of PNA and PEG. Using (99m)Tc-MAG(3)-cPNA, targeting of PNA-PA-PEG was studied in vitro and in vivo in inflammation and tumor mouse models, in both cases relying upon nonspecific diffusion for localization. In addition, cPNA-avidin was considered as a clearing agent with biotinylated PNA-PA-PEG. About 80 PNAs could be conjugated to PA provided that about 200 PEGs were also conjugated to raise the aqueous solubility of the PNA-PA-PEG polymer lowered by the addition of the PNAs. About 70% of the PNAs on this polymer in vitro either in solution or attached to beads could be successfully targeted with (99m)Tc-cPNA. In both the inflammation and tumor mouse models, between 35 and 60% of these PNAs could be targeted in the lesions. The advantage of amplification was evident when less favorable results were obtained with PNA-PA-PEG conjugated with only six PNAs. We conclude that amplification can be achieved in vivo using polymers of PNA followed by radiolabeled complementary PNA and that the application of pretargeting using polymers of PNA for amplification can improve localization.
Relatively few studies comparing different methods of labelling peptides with 99Tcm have been reported. In this investigation, we evaluated the influence of three chelators on the in vitro and in vivo properties of two small, similar peptides (HNE2 and HNE4) labelled with 99Tcm. Both peptides were labelled with hydrazinonicotinamide (HYNIC) (tricine) at pH 5-6 and with diethylenetriaminepentaacetic acid (DTPA) and mercaptoacetyltriglycine (MAG3) at both pH 5-6 and 7-8. All ten preparations were brought to pH 7.2 immediately after labelling. Each preparation labelled well and control labelling showed each label to be attached specifically at chelation sites. Analysis of 37 degrees C human serum incubates showed little evidence of label instability but high protein binding in several cases. The stability of 99Tcm to cysteine challenge for labelled DTPA- and MAG3-peptides was similar but lower than that for the HYNIC-peptides. Reverse phase HPLC of the DTPA-peptides, but not the MAG3-peptides, showed different 99Tcm species depending on labelling pH. The 3 h biodistributions in normal mice were generally independent of labelling pH for both MAG3-peptides but were heavily influenced by labelling pH for both DTPA-peptides. While significant differences in biodistribution for the same labelling method were evident between peptides, as expected, far larger differences in the case of both peptides resulted from changing chelators and, in the case of DTPA, changing the labelling method. In summary, the chelators and labelling methods influenced the biodistribution of 99Tcm in a characteristic fashion common to both peptides. Differences in biodistribution due to the different peptides were relatively small and generally lost in the much larger differences due to chelator and labelling method. In conclusion, it may be important to compare chelators and labelling methods before selecting a 99Tcm labelling method for any particular peptide.
We have shown previously that the epidermal growth factor peptide (EGF) may be radiolabeled with 99mTc at room temperature and neutral pH by using the N‐hydroxysuccinimide ester of S‐acetyl mercaptoacetyltriglycine (MAG3) as a bifunctional chelator. By a competition binding assay, we found that MAG3‐conjugated EGF retained biological activity. Furthermore, the labeled peptide exhibited saturation binding to EGF receptor‐positive tumor cell lines which could be inhibited by presaturation of the cells with unlabeled, native EGF. Biodistribution in normal mice at 3 h postadministration showed rapid clearance with minimal retention of the label in sampled organs. We have now investigated the tumor localization properties in mice of this labeled peptide. Nude mice implanted with the EGF receptor‐positive tumors A431 and LS‐174T were administered labeled EGF and a labeled control peptide (BPTI, aprotinin). Tumor uptake at 12 h postadministration was 0.44% injected dose/g for EGF/g vs. 0.09 for the control. Pretreatment of tumored mice with unlabeled EGF blocked about half the tumor uptake. Animals were also administered an anti‐EGF receptor antibody labeled with 99mTc via MAG3. Relative to the antibody, tumor‐to‐muscle ratios were improved from 6 to 15 and tumor‐to‐blood ratios from 0.4 to 7 with EGF. These favorable results along with documented evidence of overexpression of the EGF receptor in many human tumors suggest that 99mTc‐EGF should be considered further for tumor detection. © Munksgaard 1997.
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