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Tumor targeting with folic acid radioconjugates has been proposed as a promising strategy for radionuclide therapy of folate receptor α (FR)-positive cancer. Recently, it was shown that modification of radiofolates with an albumin-binding entity increased the tumor-to-kidney ratios of accumulated radioactivity in mice. The goal of this study was to evaluate the lead compound cm10 and compare it with new albumin-binding folate conjugates. Compound cm12 was designed with a long spacer consisting of a PEG-11 entity, and compound cm13 contained a short alkane chain between the albumin-binding moiety and folic acid. All of the derivatives were labeled with 177Lu (t1/2= 6.65 days, Eβ–,average= 134 keV; Eγ= 113 keV, 208 keV), a clinically established radionuclide for therapeutic purposes. The evaluation revealed that all of the albumin-binding radiofolates exhibited increased in vitro stability compared with the reference compound (177Lu-cm14) without albumin binder. Serum protein binding, determined with an ultrafiltration assay, was high (>88%) for the derivatives with albumin-binding entities. The FR-binding affinity was in the same range (KD= 4.0–7.5 nM) for all of the radiofolates, independent of the albumin-binding entity and spacer length. FR-specific uptake was proven in vitro using FR-positive KB tumor cells. In vivo studies with KB-tumor-bearing mice were performed in order to assess the tissue distribution profile of the novel radiofolates. 177Lu-cm13 showed high tumor uptake at late time points (13.3 ± 2.94% IA/g, 48 h p.i.) and tumor-to-kidney ratios (0.59 ± 0.03, 48 h p.i.) in the same range as 177Lu-cm10 (0.55 ± 0.07, 48 h p.i.). However, the tumor-to-kidney ratio of 177Lu-cm12 (0.28 ± 0.07, 48 h p.i.) was reduced compared with 177Lu-cm10 and 177Lu-cm13. The results of this study indicate that the spacer entity between folic acid and the albumin binder is of critical importance with regard to the tissue distribution profile of the radiofolate. The PEG spacer compromised the beneficial effects of the lead compound, but the design with a short alkane spacer appeared to be promising. Future studies will focus on the design of radiofolates with lipophilic and more rigid spacer entities, which may allow a further improvement of their tissue distribution profiles.

Beagle dogs are widely used in preclinical pharmacokinetic, safety, and formulation studies. However, little is known about intestinal and hepatic distribution of major enzymes and transporters involved in oral absorption and presystemic drug metabolism. We characterized mRNA levels of CYP3A12, CYP3A26, CYP2D15, UGT1A6, ABCB1(MDR1), ABCC1(MRP1), ABCG2(BCRP), SLC15A1(PEPT1), and SLC22A1(OCT1) in dog liver and along the intestine by real-time quantitative reverse transcription-polymerase chain reaction. Tissue protein levels of CYP2D15, MDR1, and PEPT1 were obtained by Western blot. Gene distribution and expression variability was statistically described by a generalized additive mixed model smoothing function and correspondence analysis. Results were compared with the expression pattern known for the human orthologs. Hepatic mRNA levels for metabolic enzymes were generally higher than those for membrane transporters, whereas in the intestine the opposite was observed. Hepatic mRNA levels followed the order CYP2D15> UGT1A6≈ CYP3A26> ABCB1≈ SLC15A1≈ SLC22A1> ABCG2> ABCC1≈ CYP3A12. Along the gut, the genes were differentially distributed with greatest expression in duodenum/upper jejunum (ABCG2), middle jejunum (ABCB1and SLC15A1), or in cecum/colon (ABCC1and CYP2D15). CYP3A12, CYP3A26, SLC22A1, and UGT1A6had a rather uniform expression. Intestinal mRNA profiles of CYP2D15, ABCB1, and SLC15A1correlated with the respective protein levels. Canine CYP3A12/26, CYP2D15, and ABCB1colonic distributions differed from those of human orthologs, whereas UGT1A6, ABCC1, ABCG2, SLC15A1, and SLC22A1were comparable to those of humans in both small and large intestine. We aim to apply these data to better interpret pharmacokinetic studies in dogs with respect to their human relevance.